Download Branched-chain amino acids alter neurobehavioral function in rats

Am J Physiol Endocrinol Metab 304: E405–E413, 2013.
First published December 18, 2012; doi:10.1152/ajpendo.00373.2012.
Branched-chain amino acids alter neurobehavioral function in rats
Anna Coppola,1,2 Brett R. Wenner,1 Olga Ilkayeva,1 Robert D. Stevens,1,3 Mauro Maggioni,4
Theodore A. Slotkin,2 Edward D. Levin,5 and Christopher B. Newgard1,2,3
1
Sarah W. Stedman Nutrition and Metabolism Center, 2Department of Pharmacology and Cancer Biology, 3Department of
Medicine, Division of Endocrinology, 4Department of Mathematics, and 5Department of Psychiatry and Behavioral Sciences,
Duke University, Durham, North Carolina
Submitted 25 July 2012; accepted in final form 11 December 2012
branched-chain amino acids; tryptophan; mood disorders; obesity
IN THE PAST FEW DECADES, changes in food consumption and
sedentary living conditions have contributed to a worldwide
obesity epidemic, including in the US, where more than 34% of
adults are obese (44, 45). Given the high prevalence of obesity and
mood disorders, there is an increasing interest in the relationship
of these two conditions (2, 17, 25, 33, 42, 58, 62) and in the
concept that overnutrition may have a direct causal link to impairment of mental function.
Recent studies have revealed that a cluster of metabolites
comprised of the branched-chain amino acids (BCAA) leucine
(Leu), isoleucine (Ile), and valine (Val) and their metabolites,
as well as the aromatic amino acids (AAA) tyrosine (Tyr) and
phenylalanine (Phe), are strongly associated with obesity and
insulin resistance in humans (1, 10, 29, 43, 67, 78). Moreover,
a very similar metabolite cluster predicts incident type 2
diabetes in longitudinal studies in humans (64) and, when
measured at baseline in obese subjects, also predicts improvement in insulin sensitivity in response to a dietary/behavioral
Address for reprint requests and other correspondence: C. B. Newgard,
Sarah W. Stedman Nutrition and Metabolism Center, Duke Independence Park
Facility, 4321 Medical Park Dr., Suite 200, Durham, NC 27704 (e-mail:
[email protected]).
http://www.ajpendo.org
weight loss intervention (63). These findings suggest the possibility that elevated BCAA could be linked to behavioral
disorders.
BCAA are transported from the blood into the central
nervous system (CNS) through the blood-brain barrier (BBB)
by the large neutral amino acid transporter 1 (LAT1) (47).
LAT1 transports all of the large neutral amino acids (LNAA),
including the BCAA and AAA, Tyr, Phe, and tryptophan (Trp).
This system is saturated at physiological amino acid concentrations, and uptake of BCAA is competitive with respect to
uptake of AAA (46). Therefore, an increase in circulating
BCAA is predicted to decrease uptake of AAA into the CNS
(19). Tyr is the precursor of norepinephrine (NE) and dopamine (DA) (37), whereas Trp is the precursor of serotonin
(5-HT) (38). Thus, the rate of production of important neurotransmitters may be affected by changes in amino acid
concentrations in the blood (18). This could potentially include
serotonin, which regulates a variety of behavioral functions,
including mood and appetite regulation (16). Recent studies
have indicated that other metabolic fates of Trp in brain could
also modulate behavior (reviewed in Ref. 39), most notably its
conversion to the neuroactive metabolite kynurenic acid
(KYNA) (30, 34, 36, 39, 81).
Several studies provide evidence that different food components have a direct impact on brain processes at many levels
(reviewed in Refs. 26, 40, and 79). Given the emergent and
tight association of BCAA and AAA with obesity and insulin
resistance, and in light of the potential behavioral impact of
AAA via their role as precursors of key neurotransmitters, we
adapted a dietary BCAA supplementation strategy as used
previously for studying the relationship of BCAA and insulin
resistance in rats (43) for studies of the impact of BCAA on
behavior. This allowed us to test the hypothesis that long-term
supplementation of BCAA leads to behavioral changes.
EXPERIMENTAL PROCEDURES
Animal studies. All procedures described in this article were approved by the Duke University Institutional Animal Care and Use
Committee. Each study involved the use of male Wistar rats (150 –
175 g; Charles River Laboratories, Durham, NC) single-caged throughout the experiment. At the end of the experiments, rats were euthanized
by decapitation and brain tissues rapidly dissected and snap-frozen in
liquid nitrogen. Blood was collected from the body trunk at the time of
euthanization.
Diets. All diets were formulated and custom made by Research
Diets. Rats were fed for 9 wk with four different diets: low-fat,
high-sucrose diet (LF); low-fat, high-sucrose diet supplemented with
Leu, Val, and Ile by 150% (LF/BCAA); high-fat diet (HF); or high-fat
diet supplemented with Leu, Val, and Ile by 150% (HF/BCAA).
Composition of each of the diets is reported in Table 1.
0193-1849/13 Copyright © 2013 the American Physiological Society
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Coppola A, Wenner BR, Ilkayeva O, Stevens RD, Maggioni M,
Slotkin TA, Levin ED, Newgard CB. Branched-chain amino
acids alter neurobehavioral function in rats. Am J Physiol Endocrinol Metab 304: E405–E413, 2013. First published December 18,
2012; doi:10.1152/ajpendo.00373.2012.—Recently, we have described a strong association of branched-chain amino acids (BCAA)
and aromatic amino acids (AAA) with obesity and insulin resistance.
In the current study, we have investigated the potential impact of
BCAA on behavioral functions. We demonstrate that supplementation
of either a high-sucrose or a high-fat diet with BCAA induces
anxiety-like behavior in rats compared with control groups fed on
unsupplemented diets. These behavioral changes are associated with a
significant decrease in the concentration of tryptophan (Trp) in brain
tissues and a consequent decrease in serotonin but no difference in
indices of serotonin synaptic function. The anxiety-like behaviors and
decreased levels of Trp in the brain of BCAA-fed rats were reversed
by supplementation of Trp in the drinking water but not by administration of fluoxetine, a selective serotonin reuptake inhibitor, suggesting that the behavioral changes are independent of the serotonergic
pathway of Trp metabolism. Instead, BCAA supplementation lowers
the brain levels of another Trp-derived metabolite, kynurenic acid,
and these levels are normalized by Trp supplementation. We conclude
that supplementation of high-energy diets with BCAA causes neurobehavioral impairment. Since BCAA are elevated spontaneously in
human obesity, our studies suggest a potential mechanism for explaining the strong association of obesity and mood disorders.
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BCAA AND BEHAVIOR
Table 1. Diet composition
Diet (kcal)
LF
LF/BCAA
HF
HF/BCAA
Protein*
Carbohydrate
Fat
Total
19
71
10
100
23
67
10
100
19
35
45
100
23
34
43
100
LF, low fat; HF, high fat; BCAA, branched-chain amino acids. All diets
were formulated and custom made by Research Diets. Low-fat/high-sucrose
diet (LF), low-fat/high-sucrose diet supplemented with leucine, valine, and
isoleucine by 150% (LF/BCAA), high-fat diet (HF), or high-fat diet supplemented with leucine, valine, and isoleucine by 150% (HF/BCAA). Data are
presented in kcal. *The protein component of the diet is 50% casein plus 50%
total amino acids, with or without 150% more branched-chain amino acids.
Fig. 1. The effect of branched-chain amino acid (BCAA)-supplemented diets on body weight and food intake. A: body weight was measured weekly throughout
the entire 9-wk study. Body weight is presented for weeks 4–9 as %change over control groups (ANOVA: not significant). B: caloric intake was measured weekly
throughout the entire 9-wk study. Caloric intake is presented for weeks 4–9 as kcal/week consumed. {ANOVA: diets [low-fat, high-sucrose diet (LF) vs. high-fat
diet (HF)], P ⬍ 0.0001}. For A and B, n ⫽ 15/group. LF/BCAA, low-fat, high-sucrose diet supplemented with leucine, valine, and isoleucine by 150%;
HF/BCAA, high-fat diet supplemented with leucine, valine, and isoleucine by 150%.
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Elevated plus maze. The elevated plus maze (EPM) test was applied to
rats as a test of anxiety based on their spontaneous avoidance of new and
open areas as a predatory risk escape behavior. Rats were fed with the
four different diets for 9 wk before being tested with the EPM. The maze
was constructed of black-painted wood with arms 55 cm long and 10.2
cm wide, standing 50.8 cm above the floor. Two opposed arms had walls
that were 15.2 cm high, and the other two opposed arms, set at 90° to the
walled arms, had railings 2 cm tall. Rats are placed in the center of the
maze facing an open arm and allowed to roam freely for a total of 300 s.
We measured the number of entries and the time spent in the open arms,
time spent in the closed arms, and the number of center crossings. An arm
entry or exit was defined as all four paws crossing the arm threshold.
The entire procedure was video recorded and analyzed with motion
detection and tracking algorithms that allow measurement of current
position and velocity of the rat as well as counting transitions between
arms and dwelling time at the center of the maze.
Fluoxetine treatment. Rats were fed with the four different diets
throughout the entire experiment. After 5 wk of diet they received
daily intraperitoneal injections of fluoxetine (10 mg·kg⫺1·day⫺1) or
saline solution for 4 wk. After the treatment, we tested the efficacy of
the antidepressant treatment by analyzing the animals with EPM.
Tryptophan supplementation. Rats were fed with the four different
diets throughout the entire experiment. After 5 wk of diet they
received regular tap water from bottles or regular tap water supplemented with tryptophan (15 mg/100 ml) from bottles for 4 wk. The
bottles were changed daily for all groups. After the treatment, we
tested the animals with EPM.
Amino acid analysis. Amino acid measurements were made by flow
injection/tandem mass spectrometry using stable isotope dilution
techniques and sample preparation methods described previously (3, 41,
76). The data were acquired using a Waters Acquity UPLC system
equipped with a triple quadrupole detector and a data system controlled
by MassLynx 4.1 MS software platform (Waters, Milford, MA).
LC-MS/MS analysis of Trp and KYNA. Tissue homogenate or
serum was filtered through Millipore Amicon Ultra-0.5 ml 3k MWCO
centrifugal filters (UFC500396). Ten microliters of isotope-labeled
internal standards containing 10 ␮M L-tryptophan-d5 (DLM-10920.5; Cambridge Isotope Laboratories) and 0.04 ␮M kynurenic3,5,6,7,8-d5 acid (D-4391; CDN Isotopes) was added to 70 ␮l of the
filtrate and to a series of calibrators. An L-tryptophan (T0254; Sigma)
calibration curve was prepared by diluting the stock solution to 0.4, 1,
2, 4, 10, and 20 ␮M, and a KYNA (K3375; Sigma) calibration curve
was prepared by diluting the stock solution to 0.0008, 0.0020, 0.0040,
0.0080, 0.0200, and 0.0400 ␮M. Tryptophan and KYNA were analyzed on a Waters Acquity UPLC system coupled to a Waters Xevo
TQ-S triple quadrupole mass spectrometer. The analytical column
(Waters Acquity UPLC HSS T3 Column, 1.8 ␮m, 2.1 ⫻ 100 mm)
equipped with a guard column (Agilent Rapid Resolution cartridge,
ZORBAX SB-C8, 3.5 ␮m, 2.1 ⫻ 30 mm) was used at 30°C, and 7.5
␮l of the sample was injected onto the column and eluted at a flow rate
of 0.3 ml/min. The gradient began with 100% eluent A (0.1% formic
acid in water) and was then programmed as follows: 0- to 2-min 0%
eluent B (95:5 acetonitrile-water, 0.1% formic acid); 2- to 10-min
gradient to 40% eluent B; 10- to 11-min gradient to 100% eluent B;
11- to 13-min hold at 100% eluent B; 13.0- to 13.5-min gradient to 0%
eluent B; 13.5- to 15.5-min hold at 100% eluent A to reequilibrate the
column. Mass transitions of m/z 205 ¡ 146 for Trp, 210 ¡ 150 for
d5-Trp, 190 ¡ 116 for KYNA, and 195 ¡ 121 for d5-KYNA were
monitored in positive ion electrospray ionization mode with the
following parameters: capillary voltage 2,000 V, cone voltage 22 V
for Trp and 2 V for KYNA, collision energy 16 V for Trp and 28 V for
KYNA. Quantitation of Trp and KYNA from raw multiple reaction
monitoring data was performed using Waters TargetLynx Quantitative
Analysis.
Monoamine analysis. The levels of monoamines and their major
metabolites were assayed using tissue homogenization and standard
HPLC-EC methods, as reported previously (52). The HPLC system
consisted of an isocratic pump (model LC1120; GBC Separations), a
Rheodyne injector (model 7725i) with a 20-␮l PEEK loop, and an
INTRO amperometric detector (Antec Leyden, Warwick, RI). The
signal was integrated using the EZChrom elite chromatography software (Scientific Software, Pleasanton, CA). The mobile phase was 50
mM H3PO4, 50 mM citric acid, 100 mg/l 1-octanesulfonic acid
(sodium salt), 40 mg/l EDTA (disodium salt dihydrate), 2 mM KCl,
and 3% methanol, corrected to pH 3.0 with NaOH. The mobile phase
was continually degassed with a Degasys Populaire on-line degasser
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BCAA AND BEHAVIOR
Fig. 2. The effect of BCAA-supplemented diets
on behavior. A: time spent in the open arms of
the elevated plus maze during five 5-min sessions of the test. Time is expressed in seconds
(ANOVA: BCAA, P ⬍ 0.0002). B: no. of closedarm entries during 5-min sessions of the test
as index of motor activity (ANOVA: not
significant). Data are presented as means ⫾ SE
(n ⫽ 15/group).
RESULTS
Long-term consumption of diets supplemented with BCAA
affects behavior. To investigate the consequence of overnutrition and meal-based supplementation of BCAA on stress and
behavior, we fed young Wistar rats with four different highenergy diets for a period of 9 wk: LF, LF/BCAA, HF, or
HF/BCAA (see Table 1 for diet composition). Food intake by
weight of food consumed and in terms of caloric intake was
higher in rats fed the HF-based compared with the LF-based
diets (P ⬍ 0.0001), but no significant differences were observed in food intake in groups fed with HF compared with
HF/BCAA or LF compared with LF/BCAA diets (Fig. 1B).
This suggests that supplementation of the diets with BCAA did
not affect palatability of the food or cause a taste aversion
response. There was a trend to increase body weight more
rapidly in rats fed with HF diet compared with LF, but the
differences were not statistically significant (Fig. 1A). Similarly, supplementation of either diet with BCAA caused a trend
for a lower rate of body weight gain relative to the unsupplemented diets, but these effects also did not reach statistical
significance. Note that we have shown previously that feeding
of either the HF or LF diets increases weight gain relative to a
low-fat, low-sucrose, standard chow diet (43, 57).
We analyzed the impact of 9 wk of feeding of the various
diets on behavior of the rats using the EPM test. We found that
BCAA supplementation (both LF/BCAA and HF/BCAA) induced a significant reduction of the time spent in the open
arms, which was suggestive of an anxiogenic effect of BCAA
supplementation leading to less exploratory behavior (Fig. 2A).
The behavior of the rats fed the LF diet was indistinguishable
from the behavior of rats fed the HF diet, and in fact, a
comparison of all diets identified only a main effect of BCAA
(P ⬍ 0.002) with no diet (LF vs. HF) ⫻ BCAA interaction.
Because differences in general motor activity among groups
could have influenced the exploratory behavior in the EPM, we
measured the number of closed-arm entries as an index of
general ambulatory activity (12, 54). No diet-induced difference
was found in overall ambulatory activity of the rats (Fig. 2B). We
found that the effect of dietary supplementation of BCAA on
behavior required long-term consumption of the diets, as overfeeding for only 2 wk did not induce significant changes (data
not shown). We also found no significant differences in the
levels of the stress hormone corticosterone in serum samples
Fig. 3. The ratio of plasma levels of single
amino acids to plasma levels of the sum of
all large neutral amino acids (冱LNAA). The
increased valine/冱LNAA and leucine/isoleucine/冱LNAA ratios predict increased uptake through the blood-brain barrier (BBB).
The decreased tyrosine/冱LNAA and tryptophan/冱LNAA ratios predict decreased uptake
through the BBB (ANOVA: BCAA, P ⬍
0.0001 for all 4 ratios). Data are presented as
means ⫾ SE (n ⫽ 5/group).
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(Sanwa Tsusho, Tokyo, Japan) and delivered at a flow rate of 0.26
ml/min.
Binding capacity assay. The assay methodologies used in this study
have appeared previously (65), so only brief descriptions will be
provided here. 5-HT1A receptors, 5HT2 receptors, and the 5-HT
transporter were studied. Frontal cortex tissue was homogenized
(Polytron; Brinkmann Instruments, Westbury, NY) in ice-cold 50 mM
Tris (pH 7.4), and the homogenates were sedimented at 40,000 g for
15 min. The pellets were washed, resedimented, and dispersed. Two
radioligands were used to determine 5-HT receptor binding: 1 nM
[3H]8-hydroxy-2-(di-n-propylamino)tetralin (specific activity, 170.2
Ci/mmol; Perkin-Elmer Life Sciences, Boston, MA;) for 5-HT1A
receptors and 0.4 nM [3H]ketanserin (specific activity, 67 Ci/mmol;
Perkin-Elmer) for 5-HT2 receptors. For 5-HT1A receptors, incubations lasted for 30 min at 25°C in a buffer consisting of 50 mM Tris
(pH 8), 2 mM MgCl2, and 2 mM sodium ascorbate; 100 ␮M 5-HT
(Sigma) was used to displace specific binding. For 5-HT2 receptors,
incubations lasted 15 min at 37°C in 50 mM Tris (pH 7.4), and
specific binding was displaced with 10 ␮M methylsergide (Sandoz
Pharmaceuticals, East Hanover, NJ). Incubations were stopped by the
addition of a large excess of ice-cold buffer, and the labeled membranes were trapped by rapid vacuum filtration onto glass fiber filters
that were presoaked in 0.15% polyethyleneimine (Sigma), and following several washes, radiolabel retained on the filter was measured
by scintillation counting. For binding to the presynaptic 5-HT transporter, the membrane suspension was incubated with 85 pM [3H]paroxetine (Perkin-Elmer; specific activity 24.4 Ci/mmol) with or
without addition of 100 ␮M 5-HT to displace specific binding, and
incubations lasted 120 min at 20°C. Binding was calculated relative to
membrane protein.
Statistical analysis. Data were assessed using multivariate ANOVA
with factors of diet (LF vs. HF) and BCAA supplementation. When
the rats were treated with fluoxetine or Trp, we included these
additional factors. Significance was assumed at P ⬍ 0.05.
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BCAA AND BEHAVIOR
Fig. 4. The effect of BCAA-supplemented diets
on levels of aromatic amino acids in prefrontal
cortex. A: levels of tyrosine (ANOVA: BCAA,
P ⬍ 0.0001). B: level of tryptophan (ANOVA:
BCAA, P ⬍ 0.0002) in prefrontal cortex in rats
fed the indicated diets. Data are presented as
means ⫾ SE (n ⫽ 5/group).
BCAA-supplemented diets affect 5-HT transport in the CNS
but not binding capacity of 5-HT receptors. Our results for
5-HT levels and turnover suggested that release of 5-HT into
the synapse was maintained despite reductions in 5-HT levels
caused by BCAA supplementation. To assess the functional
measures that would verify this interpretation, we measured the
binding capacity of the 5-HT1A and 5-HT2 receptors as well as
the 5-HT transporter. Reduced 5-HT synaptic communication
would be expected to upregulate 5-HT receptors, but we found
no effect of BCAA supplementation on the binding capacity of
either of the receptor subtypes. In contrast, we did find a
significant decrease in the binding capacity of the 5-HT transporter in rats fed the BCAA-supplemented diet compared with
control (Fig. 6); reduced presynaptic recapture would explain
the maintenance of 5-HIAA levels in the face of reduced 5-HT.
Lack of effect of the antidepressant fluoxetine on BCAAinduced behavioral changes. We tested the impact of a 5-HT
reuptake inhibitor, fluoxetine, on the BCAA-induced changes
in behavior in the EPM test. Rats were fed the four different
diets for 5 wk and then continued on those diets for an
additional 4 wk with or without fluoxetine treatment. Whereas
rats fed the BCAA-supplemented diets spent less time in the
open arms in the EPM, confirming the findings of Fig. 1,
fluoxetine treatment did not improve performance in rats fed
the BCAA-supplemented diets (Fig. 7). Taken together, data in
Figs. 6 and 7 suggest that, although BCAA supplementation
alters presynaptic 5-HT levels, functional activity is maintained as a consequence of increased release and reduced
recapture of the transmitter so that the anxiogenic effect of
BCAA is not dependent on the changes in 5-HT function.
Tryptophan supplementation rescues the stressed behavior
in rats fed BCAA-supplemented diets and lowers brain KYNA
levels. In an attempt to modify the concentration of LNAA in the
circulation and hence, to restore the uptake of Trp through the
BBB, we supplemented rats fed the various diets with Trp dissolved in drinking water. Trp supplementation increased the level
of Trp in the circulation in all groups of animals (main effect of
Fig. 5. The effect of BCAA-supplemented diets
on levels of serotonin (5-HT) and its metabolites in the central nervous system. A: levels of
5-HT measured in prefrontal cortex (ANOVA:
BCAA, P ⬍ 0.003). B: 5-HT major metabolite
5-HIAA in prefrontal cortex (ANOVA: not
significant). C: 5-HT turnover rate calculated
as the ratio of 5-HT and 5-HIAA (ANOVA:
BCAA, P ⬍ 0.004). Data are presented as
means ⫾ SE (n ⫽ 5/group).
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collected from the different dietary groups at the time of
euthanization (data not shown).
AAA and serotonin levels in the CNS are reduced by BCAA
supplementation. To determine whether the increase of BCAA
in the diet may be responsible for disrupted transport of AAA
across the BBB, we measured amino acid levels in plasma and
prefrontal cortex using tandem mass spectrometry. We found a
clear increase in the ratio of BCAA/冱LNAA (molar sum of
LNAA) in plasma of LF/BCAA- and HF/BCAA-fed rats relative to LF and HF groups, respectively (Fig. 3). This change
in ratio predicts an increase in the central uptake of BCAA
through the BBB. Conversely, we found a decrease in the ratio
of AAA/冱LNAA in plasma that predicts reduction in AAA
uptake into the CNS (18). Consistent with these predictions, we
observed a decreased concentration of Tyr (Fig. 4A) and Trp
(Fig. 4B) in the brain tissues of rats in both the HF/BCAA and
LF/BCAA groups compared with their control groups with no
BCAA supplementation. Concerning both the serum and brain
amino acid ratio values, a main effect of BCAA supplementation was seen, with no diet ⫻ BCAA supplementation interaction.
To test whether dietary supplementation of BCAA perturbs
brain monoamine pathways, we measured 5-HT, NE, and DA
levels as well as the major metabolites of 5-HT and DA
(5-hydroxyindolacetic acid, 3,4-dihydroxyphenylglycol, and
3,4-dihydroxyphenylacetic acid, respectively) in prefrontal
cortex. We found a significant decrease in 5-HT concentration
in BCAA-fed rats compared with their control groups. Nevertheless, the concentration of 5-HIAA, which reflects the
amount of 5-HT released into the synapse, was unchanged.
Consequently, the turnover rate of serotonin, measured as the
5-HIAA/5-HT ratio, was elevated significantly in the BCAA
groups, reflecting compensatory hyperactivity that offset the
5-HT deficits (Fig. 5). The changes were selective for serotonergic systems, since we did not find significant BCAAinduced changes for NE and DA in the frontal cortex (data not
shown).
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BCAA AND BEHAVIOR
Fig. 6. The effect of BCAA-supplemented
diets on the binding capacity of selected 5-HT
receptors in the brain. A: 5-HT1A receptor
(ANOVA: not significant). B: 5-HT2 receptor
(ANOVA: not significant). C: 5-HT transporter (5-HTT) (ANOVA: BCAA, P ⬍ 0.04;
C), all measured in prefrontal cortex. Data are
presented as means ⫾ SE (n ⫽ 5/group).
Fig. 7. The effect of the 5-HT reuptake inhibitor fluoxetine on the behavior of
BCAA-supplemented rats. Time spent in the open arms of the elevated plus maze
during five 5-min sessions of the behavioral test following 4 wk of fluoxetine
administration. Time is expressed in seconds (ANOVA: BCAA, P ⬍ 0.04, fluoxetine;
not significant). Data are presented as means ⫾ SE (n ⫽ 5/group).
an endogenous neuroactive metabolite of Trp, in the frontal
cortex. We found a decrease in KYNA levels after supplementation of BCAA in both the LF and HF diet backgrounds (P ⬍
0.0001 for BCAA effect). Mirroring our findings with brain
Trp levels, supplementation of Trp in the drinking water
restored KYNA levels in LF/BCAA and HF/BCAA-fed rats to
the levels found in LF- and HF-fed controls (P ⬍ 0.0001 for
Trp effect; Fig. 9B).
Importantly, Trp addition to the drinking water increased the
time spent in open arms (main effect of Trp), regardless of
whether animals received BCAA supplementation or not. The
positive effect of Trp offset the reduction caused by BCAA
supplementation so that BCAA-treated animals given Trp showed
a behavioral pattern similar to that of the non-BCAAsupplemented groups without Trp (compare LF-water with LF/
BCAA-Trp; compare HF-water with HF/BCAA-Trp; Fig. 10).
DISCUSSION
As the prevalence of obesity has grown, a clear relationship
between obesity and mental illness has emerged (17, 55). Both
obesity and mood disorders are associated with diabetes (4),
cardiovascular diseases (50), and increased risk of mortality
(11, 60). Treatment of obesity often leads to a decrease in
depression (74), the most striking example being weight loss
after gastric bypass surgery (15). Taken together, these studies
support the idea of a common pathophysiology between obesity and mental illness, but much remains unknown about
underlying mechanisms.
In the current study, we have built upon recent findings of
strong associations between the levels of BCAA and AAA,
obesity, and obesity-related metabolic dysregulation (10, 43,
63) and have begun to explore the possible connection between
BCAA, AAA, and behavioral modifications. We have found
that dietary supplementation of BCAA in either of two energydense diets, a low-fat, high-sucrose diet or a high-fat diet, has
a clear effect to decrease exploratory behavior in rats subjected
to an EPM test, indicating increased stress/anxiety (49, 53).
Importantly, whereas this effect was evident in response to
BCAA supplementation, changing the diet from low fat, high
sucrose to high fat, low sucrose had no comparable effect. This
provides important evidence that specific dietary components,
rather than fat or caloric content, can contribute to diet-induced
changes in behavior.
One likely mechanism for the BCAA effect is the impact of
increased BCAA on transport of competing amino acids across
the BBB into the CNS. Synthesis of several neurotransmitters
is dependent upon the transport of AAA precursors to maintain
synaptic levels and activity, notably 5-HT (synthesized from
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tryptophan; Fig. 8A), but the increase in Trp was smaller in
BCAA-supplemented rats compared with their control groups
(BCAA ⫻ Trp interaction, P ⬍ 0.03; P ⬍ 0.04 for Trp effect in
the BCAA group, P ⬍ 0.001 for Trp effect in the control group).
Nevertheless, Trp supplementation restored the Trp/冱LNAA ratio
in LF/BCAA- and HF/BCAA-fed rats to the levels found in LFand HF-fed controls (no significant BCAA ⫻ Trp interaction; Fig.
8B), most likely because the affinity of Trp for the transporter
LAT1 is higher than its affinity for any of the BCAA (48, 66). As
predicted by the Trp ratio, Trp levels in brain tissues were
increased in rats fed BCAA-supplemented diets ⫹ Trp in the
drinking water compared with the rats fed BCAA-supplemented
diets and drinking regular tap water (Fig. 9A). The increases in
both plasma (Fig. 8A) and brain Trp levels induced by Trp
addition to the drinking water were smaller in BCAAsupplemented rats compared with their control groups, although
the difference was at the margin of significance (P ⬍ 0.07 for the
BCAA ⫻ Trp interaction). Considering that the measurements of
Trp in the brain are dependent on the plasma levels, we conducted
an analysis comparing treatment effects in plasma and brain and
found an overall interaction for BCAA ⫻ Trp (P ⬍ 0.02) that was
indistinguishable between brain and plasma (no BCAA ⫻ Trp ⫻
tissue interaction), indicating that the effects in the brain do
actually reflect the same BCAA ⫻ Trp interaction as that found in
the plasma.
To further support our hypothesis that BCAA-supplemented
diets disrupt Trp uptake and metabolism, we measured KYNA,
E410
BCAA AND BEHAVIOR
Fig. 8. The effect of 4 wk of tryptophan (Trp)
supplementation on plasma Trp and Trp/
冱LNAA ratio. Supplementation of Trp in the
drinking water of BCAA-fed rats restores
plasma Trp to the levels found in control animals fed unsupplemented diets. A: levels of
Trp in the plasma (ANOVA: BCAA, P ⬍
0.0002; Trp, P ⬍ 0.0001; BCAA ⫻ Trp, P ⬍
0.03). B: plasma Trp levels shown as ratio of
Trp to the sum of the serum level of all LNAA
(Trp/冱LNAA) (ANOVA: BCAA, P ⬍ 0.004;
Trp, P ⬍ 0.0001; BCAA ⫻ Trp; not significant). Data are presented as means ⫾ SE (n ⫽
15/group).
release and reduction in recapture. We confirmed this interpretation by showing that the behavioral effects of BCAA were
not reversed by fluoxetine, a serotonin-specific reuptake inhibitor that boosts 5-HT levels in the synapse.
Several studies have shown an association of low serotonin
levels with the increase in anxious-like behavior (7, 14, 68).
Most of these previous studies assessed the effect of short-term
intervention as opposed to our long-term (9 wk) feeding
regimen. Our dietary manipulation affected the performance of
the rats only when tested with the EPM, but not when behavior
was evaluated with open-field or sucrose preference tests (data
not shown). Most of the prior studies did not use EPM, and
different tests may reveal different elements of anxious or
depressed behaviors (22, 51).
We demonstrate that 4 wk of Trp supplementation is sufficient to reverse cautious behavior in rats fed BCAA-supplemented diets. We show that Trp supplemented into the drinking water increased the level of this amino acid in the circulation of rats fed BCAA-supplemented diets and increased
uptake of Trp through the BBB. This is consistent with several
clinical studies showing the positive effect of Trp supplementation on mood disorders (reviewed in Ref. 71). Our data in
aggregate demonstrate that the behavioral effects by BCAA
supplementation of high-energy diets are not explained by
modification of the serotoninergic pathway.
Further studies will be needed to define the mechanism of
the anxiogenic effects of chronic BCAA supplementation in
rats. One possible mechanism is dysregulation of the kynurenine (KYN) pathway secondary to reduced Trp levels. Only
5% of the Trp in the brain is converted to serotonin, with most
of the remainder being catabolized through the KYN pathway
(24). KYN can either be metabolized to quinolinic acid
(QUIN) or undergo transamination to KYNA. The kynurenine
metabolites have a central effect, and recently, there has been
Fig. 9. The effect of Trp supplementation on
brain Trp and kynurenic acid levels. A: Trp
levels in prefrontal cortex following 4 wk of
Trp supplementation. (ANOVA: BCAA, P ⬍
0.002; Trp, P ⬍ 0.0001; BCAA ⫻ Trp, P ⫽
0.07). Data are presented as %change over
control groups (n ⫽ 15/group). B: kynurenic
acid levels measured in prefrontal cortex following 4 wk of Trp supplementation (ANOVA:
BCAA, P ⬍ 0.0001; Trp, P ⬍ 0.0001; no
effect for BCAA ⫻ Trp interaction). Data are
presented as pmol/mg of tissue (n ⫽ 15/
group).
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Trp), NE, and DA (synthesized from Tyr). Both BCAA and
AAA engage with the large neutral amino acid transporter (18,
19), so a rise in BCAA would be predicted to cause a decrease
in transport of the competing AAA. Consistent with this
prediction, we found a decrease in Trp and Tyr concentrations
in the brain in BCAA-supplemented rats.
A key question was whether the reduced AAA availability
was sufficient to have an impact on transmitter levels or
synaptic function. The measured decrease in brain Tyr levels
did not result in a corresponding change in either NE or DA,
likely because tyrosine hydroxylase, the rate-limiting enzyme
in the generation of NE and DA, is close to full saturation with
the tyrosine substrate, so changes in availability do not alter the
net biosynthetic capability (31). Indeed, prior studies show
only a limited sensitivity of NE and DA to circulating Tyr
levels (8, 20). In contrast, tryptophan hydroxylase, the ratelimiting enzyme in the synthetic pathway of 5-HT, is subsaturated at normal levels of Trp, the AAA precursor for this
transmitter (9, 77). Therefore, diminished substrate availability
does have the potential to decrease 5-HT synthesis and affect
5-HT-related behaviors (67, 72, 80). Here, we found a decrease
in CNS 5-HT levels in animals fed on the BCAA-supplemented diets. However, the levels of 5-HIAA, which reflect the
amount of 5-HT released into the synapse, were unchanged,
indicating that compensatory mechanisms were activated to
offset the reduction in 5-HT availability. In keeping with this
interpretation, 5-HT receptor binding was unchanged, whereas
if presynaptic function had been impaired, binding sites would
have been upregulated. We identified the underlying compensatory mechanisms: increased presynaptic activity (higher neurotransmitter turnover) and reduced synaptic recapture (decreased 5-HT transporter binding). These results suggest that,
although the amount of 5-HT is reduced by BCAA, net serotonergic function was maintained by compensatory increases in
BCAA AND BEHAVIOR
E411
strong association between obesity and behavioral abnormalities, including depression and anxiety.
ACKNOWLEDGMENTS
We thank Dr. Cynthia Kuhn, Dr. William C. Wetsel, and Dr. Olga
Temofeeva for their insightful advice and input in the behavioral studies. We
are grateful to Dr. Frederic Seidler for assistance in the binding assays.
GRANTS
The studies were supported by National Institutes of Health (NIH) Grant
PO1-DK-58398 and a Freedom to Discover Award in Metabolic Research
from Bristol-Meyers Squibb (to C. B. Newgard). A. Coppola is grateful for
salary support from NIH-sponsored Duke Endocrine Training Grant 5T32DK-007012-35.
DISCLOSURES
increasing interest in their involvement in the etiology of mood
disorders (35, 36, 39, 70) and as targets for new drugs (56, 61).
In particular, KYNA is an antagonist of the glycine-binding
site of the NMDA receptor (6, 21) and may serve as an
endogenous antiexcitotoxic agent by modulating glutaminergic
neurotransmition to achieve neuroprotective effects (5, 28).
Whereas KYN and QUIN exerted an endogenous anxiogenic
effect in experimental models (35, 73), KYNA has an anxiolytic pharmacological profile when tested in the EPM model
(34, 35). Therefore, our findings of a decrease in KYNA and
Trp in response to BCAA supplementation are consistent with
a model in which the fall in KYNA decreases the level of an
anxiolytic agent. Moreover, our findings of reversal of BCAAinduced behavioral abnormalities by Trp supplementation,
coupled with normalization of KYNA and Trp levels and the
absence of an effect of fluoxetine, are consistent with a serotonin-independent mechanism of BCAA-induced behavioral
change that could involve KYN pathway metabolites.
Mood disorders are complex and likely involve an intricate
interplay between a variety of factors. The contribution of
monoamines to the pathophysiology of mood disorders was
uncovered in the 1950s with the empirical discovery of drugs
that were able to induce or to cure depression by altering the
levels of NE and 5-HT (23, 59). Despite the progress in our
understanding of the neurobiology of the brain, we still have an
incomplete picture of the pathophysiology of depressive disorders, and monoamine-based drugs are still the most commonly prescribed therapeutic agents. Emerging insights from
neurobiological studies suggest that the etiology of mood
disorders is likely to involve dysregulation of neural plasticity
(13, 32, 69). This hypothesis is consistent with our data
showing changes in animal behavior only after a long-term
dietary manipulation.
To summarize, the long-term consumption of a diet rich in
BCAA disrupted the transport of Trp across the BBB in rats,
leading to reduced exploratory behavior of rats in EPM testing,
a sign of increased anxiety. Recent studies demonstrating a
strong association between BCAA levels, obesity, and obesityrelated metabolic disorders (27, 29, 43, 62, 63, 74), when
linked to the findings reported here, may help to explain the
No conflicts of interest, financial or otherwise, are declared by the authors.
AUTHOR CONTRIBUTIONS
A.C., B.R.W., T.A.S., E.D.L., and C.B.N. contributed to the conception and
design of the research; A.C., B.R.W., O.I., R.D.S., and E.D.L. performed the
experiments; A.C., B.R.W., O.I., R.D.S., M.M., T.A.S., E.D.L., and C.B.N.
analyzed the data; A.C., B.R.W., O.I., R.D.S., T.A.S., E.D.L., and C.B.N.
interpreted the results of the experiments; A.C. prepared the figures; A.C.
drafted the manuscript; A.C., B.R.W., O.I., R.D.S., M.M., T.A.S., E.D.L., and
C.B.N. approved the final version of the manuscript; T.A.S., E.D.L., and
C.B.N. edited and revised the manuscript.
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