Am J Physiol Regul Integr Comp Physiol 307: R310–R320, 2014.
First published June 4, 2014; doi:10.1152/ajpregu.00116.2014.
Leucine acts in the brain to suppress food intake but does not function as a
physiological signal of low dietary protein
Thomas Laeger,1 Scott D. Reed,1 Tara M. Henagan,1 Denise H. Fernandez,1 Marzieh Taghavi,2
Adele Addington,2 Heike Münzberg,1 Roy J. Martin,1 Susan M. Hutson,2 and Christopher D. Morrison1
1
Pennington Biomedical Research Center, Baton Rouge, Lousiana; and 2Virginia Polytechnic Institute and State University,
Blacksburg, Virginia
Submitted 17 March 2014; accepted in final form 31 May 2014
Laeger T, Reed SD, Henagan TM, Fernandez DH, Taghavi M,
Addington A, Münzberg H, Martin RJ, Hutson SM, Morrison
CD. Leucine acts in the brain to suppress food intake but does not
function as a physiological signal of low dietary protein. Am J Physiol
Regul Integr Comp Physiol 307: R310 –R320, 2014. First published
June 4, 2014; doi:10.1152/ajpregu.00116.2014.—Intracerebroventricular injections of leucine are sufficient to suppress food intake, but it
remains unclear whether brain leucine signaling represents a physiological signal of protein balance. We tested whether variations in
dietary and circulating levels of leucine, or all three branched-chain
amino acids (BCAAs), contribute to the detection of reduced dietary
protein. Of the essential amino acids (EAAs) tested, only intracerebroventricular injection of leucine (10 ␮g) was sufficient to suppress
food intake. Isocaloric low- (9% protein energy; LP) or normal- (18%
protein energy) protein diets induced a divergence in food intake, with
an increased consumption of LP beginning on day 2 and persisting
throughout the study (P ⬍ 0.05). Circulating BCAA levels were
reduced the day after LP diet exposure, but levels subsequently
increased and normalized by day 4, despite persistent hyperphagia.
Brain BCAA levels as measured by microdialysis on day 2 of diet
exposure were reduced in LP rats, but this effect was most prominent
postprandially. Despite these diet-induced changes in BCAA levels,
reducing dietary leucine or total BCAAs independently from total
protein was neither necessary nor sufficient to induce hyperphagia,
while chronic infusion of EAAs into the brain of LP rats failed to
consistently block LP-induced hyperphagia. Collectively, these data
suggest that circulating BCAAs are transiently reduced by dietary
protein restriction, but variations in dietary or brain BCAAs alone do
not explain the hyperphagia induced by a low-protein diet.
branched-chain amino acids; protein restriction; hypothalamus; macronutrient; food intake
ALTHOUGH THE STUDY OF ingestive behavior historically has
focused largely on the regulation of energy homeostasis, the
consumption of adequate amounts of protein, specifically essential amino acids (EAAs), is also central to health and
survival. Therefore, it seems likely that physiological systems
exist to ensure sufficient protein intake. Alterations in dietary
protein content can have profound effects on food intake, with
diets high in protein suppressing food intake and diets moderately low in protein increasing food intake (2, 11, 18, 23, 38,
39). Furthermore, when given the choice between diets that
differ in protein content, many species will self-select between
diets to ensure adequate consumption of protein (16, 24, 27),
often at the expense of carbohydrate and fat (9, 19, 31).
Address for reprint requests and other correspondence: C. D. Morrison,
Pennington Biomedical Research Center, 6400 Perkins Rd., Baton Rouge, LA
70808 (e-mail: morriscd@pbrc.edu).
R310
Despite these behavioral observations, the mechanism regulating protein intake is largely unknown (21). Recent work
has focused on the branched-chain amino acid (BCAA) leucine
as a potential protein signal. Intracerebroventricular injections
of leucine suppress food intake and regulate key signaling
systems (mTOR/AMPK) within hypothalamic neurons, while
increased dietary leucine content reproduces the anorectic
effects of a high-protein diet (3, 5, 23, 29). While these data
demonstrate that administration of excess leucine either to the
diet or the brain is sufficient to suppress food intake, it remains
unclear whether physiological fluctuations in circulating or
brain leucine actually contribute to the regulation of dietary
protein intake or selection. Here, we use the adaptive hyperphagia that occurs in response to a low-protein diet to test
the physiological role of dietary leucine and BCAAs in the
detection of protein restriction.
MATERIALS AND METHODS
Animals and diets. Male Sprague-Dawley (SD) rats (5–7 wk of age;
Charles River Laboratories, Wilmington, MA) were used in all experiments. Rats were adapted to single housing in standard shoe-box
cages with a 12:12-h light-dark cycle for at least 7 days before the
study. Standard rodent chow (4.1 kcal/g, 28.5% of energy from
protein, 13.5% of energy from fat, 58% of energy from carbohydrate;
no. 5001, LabDiet, St. Louis, MO) was provided ad libitum unless
otherwise noted. Experimental diets (seven variations in total protein,
leucine, or BCAA content) were formulated and produced by Research Diets (New Brunswick, NJ) using casein and/or synthetic
amino acids as the protein source (Table 1). Diets were made isocaloric via concurrent changes in carbohydrate content, while maintaining a constant fat content. The control (20% casein) and low-protein
(10% casein) diets were chosen to maintain consistency with previous
work from our laboratory and the laboratory of Dr. Roy Martin (23,
37, 38). These previous experiments demonstrate that a 10% casein
diet reliably produces hyperphagia in rats, and the goal of the current
work was to determine whether reductions in leucine in the diet or
brain contribute to this hyperphagia. All experiments were approved
by the Institutional Animal Care and Use Committee at Pennington
Biomedical Research Center.
Intracerebroventricular surgery. For cannulation of the third ventricle (22, 40), rats were anesthetized via chamber induction of 3%
isoflurane in oxygen and were maintained on 3% isoflurane in oxygen
via a non-rebreathing mask. The dorsal cranium was prepped for
aseptic surgery, and a dorsal midline incision extending from the
frontal sinuses to just caudal to the occipital protuberance was retracted laterally, allowing blunt dissection and debridement of the dorsal
subcutaneous fascia and connective tissue. Bone screws (0 – 80 ⫻ 1/8,
Plastics One; Roanoke, VA) were placed surrounding the cannulation
site to serve as anchors for the methacrylate placed later. A 22-gauge
stainless-steel cannula (Plastics One) or CMA 11 guide cannula
(CMA Microdialysis, Kista, Sweden) was implanted after drilling
(engraving cutter, 0.8-mm diameter; Dremel, Racine, WI), an entry
0363-6119/14 Copyright © 2014 the American Physiological Society
http://www.ajpregu.org
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BCAAS AND THE RESPONSE TO PROTEIN RESTRICTION
Table 1. Diet formulations
Product/Ingredient, g
Casein
L-Cystine
L-Isoleucine
L-Leucine
L-Valine
L-Lysine
L-Methionine
L-Phenylalanine
L-Threonine
L-Tryptophan
L-Histidine
L-Alanine
L-Arginine
L-Aspartic acid
L-Glutamic acid
Glycine
L-Proline
L-Serine
L-Tyrosine
Total AAs
Corn starch
Maltodextrin 10
Sucrose
Cellulose
Soybean oil
Lard
Mineral mix S10022C
Calcium carbonate
Calcium phosphate
Potassium citrate
Potassium phosphate
Sodium chloride
Vitamin mix V10037
Choline bitartrate
Total
kcal/gram
% of E by protein
% of E by fat
% of E by carbohydrate
D11051802 10%
(LP) casein
D11051801 20%
(NP) casein
D11051804
LP⫹All
D11051805
NP-LEU
D11051806
LP⫹LEU
D11051807
NP-BCAA
D11051808
LP⫹BCAA
100
1.5
200
3
1.5
440
150
107
50
25
75
3.5
10.0
3.5
2.5
6.9
2.6
10
2.5
990.4
4.1
9
22
69
3
376
125
107
50
25
75
3.5
12.5
0
2.5
6.9
2.6
10
2.5
1001.3
4.1
18
22
60
100
3.6
3.8
7.9
4.7
6.6
2.6
4.2
3.6
1.1
2.3
2.6
3
6.1
19.1
1.5
8.9
5
4.6
91.2
376
125
107
50
25
75
3.5
10.0
3.5
2.5
6.9
2.6
10
2.5
990.5
4.1
18
22
60
100
3.6
3.8
0
4.7
6.6
2.6
4.2
3.6
1.1
2.3
2.6
3
6.1
19.1
1.5
8.9
5
4.6
83.3
384
125
107
50
25
75
3.5
10.0
3.5
2.5
6.9
2.6
10
2.5
990.5
4.1
17
22
61
100
1.5
0
7.9
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
9.4
432
150
107
50
25
75
3.5
10.0
3.5
2.5
6.9
2.6
10
2.5
990.4
4.1
10
22
68
100
3.6
0
0
0
6.6
2.6
4.2
3.6
1.1
2.3
2.6
3
6.1
19.1
1.5
8.9
5
4.6
74.8
392
125
107
50
25
75
3.5
10.0
3.5
2.5
6.9
2.6
10
2.5
990.5
4.1
16
22
62
100
1.5
3.8
7.9
4.7
0
0
0
0
0
0
0
0
0
0
0
0
0
0
17.9
424
150
107
50
25
75
3.5
10.0
3.5
2.5
6.9
2.6
10
2.5
990.4
4.1
10
22
68
LP, low protein; NP, normal protein, LEU, leucine; BCAA, branched-chain amino acid; E, energy.
site at coordinates ⫺2.2 mm (Y) from bregma and ⫺7.5 mm (Z) from
the skull surface. Dental methacrylate was placed around the cannula
and extended to anchor points at the previously placed bone screws.
A 28-gauge obdurator or dummy was inserted in the cannula lumen,
and the exposed skull was covered by partially closing the skin
incision. Cannulation of the lateral ventricle followed an identical
procedure, except for the cannula-targeted coordinates [⫺0.9 mm (X),
⫺1.5 mm (Y) from bregma; ⫺3.0 mm (Z) from the skull surface]. All
rats received 5 mg/kg body wt carprofen (Pfizer, New York, NY)
subcutaneously preoperatively and 24 h postoperatively as an analgesic. Before further study, all rats were given 1 wk to recover from
surgery. During this time, food intake, body weight, and behavior
were observed daily. Rats that failed to regain body weight postsurgery were excluded from the experiment. To verify correct cannula
placement, an ink solution was injected through the cannula at
euthanasia to confirm correct localization to the ventricle. The third
ventricle was selected for acute intracerebroventricular amino acid
injection because leucine was previously shown (23) to reduce food
intake following third ventricular application. The lateral ventricle
was chosen for chronic intracerebroventricular amino acid infusion
for two reasons: 1) the lateral ventricle is a large structure to accommodate the constant flow of fluid and large amino acid doses, and
2) because the cerebrospinal fluid (CSF) flows from lateral via third to the
fourth ventricle, amino acid infusion into the lateral ventricle maxi-
mizes the potential exposure of the brain to amino acids, while also
delivering amino acids into the third ventricle. Finally, surgical
implantation of a microdialysis guide cannula into the third ventricle
was performed, as described above, and animals were given at least 1
wk to recover from surgery.
Experiment 1: effects of normal and LP diets on food intake. To
confirm low-protein-induced hyperphagia, rats (7–9/group) maintained on a chow diet were transitioned (during the mid-light cycle) to
either a control, normal-protein diet (NP, 20% casein) or a low-protein
diet (LP, 10% casein) diet, and daily food intake was recorded for 7
days. After 7 days on the diet, NP rats weighed 209.3 ⫾ 4.0 g, while
LP rats weighed 199.6 ⫾ 3.5 g (means ⫾ SE).
Experiment 2: comparative effects of select amino acids on food
intake. To determine whether leucine is unique in its capacity to
regulate food intake, multiple cohorts of SD rats were implanted with
indwelling third ventricle cannulas, as described above. Rats were
maintained on standard rodent chow and were fasted 24 h prior to
injection. Individual amino acids were injected as a single bolus (10
␮g, 2 ␮l; 15–20 rats/group) 2 h prior to lights off, and 24-h intake of
standard rodent chow was assessed. The rats were given at least 1 wk
to recover between injections and were given a maximum of three
injections. Average body weights at the time of injection ranged from
240 to 325 g. To test whether leucine was sufficient to suppress intake
in rats consuming a LP diet, a group of rats were implanted with an
AJP-Regul Integr Comp Physiol • doi:10.1152/ajpregu.00116.2014 • www.ajpregu.org
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BCAAS AND THE RESPONSE TO PROTEIN RESTRICTION
intracerebroventricular cannula, and after 7 days of recovery, they
were placed on LP diet for 4 days. Rats were injected intracerebroventricularly with either saline or leucine (10 ␮g, 2 ␮l; 10/group) in the
fed state, and 24-h food intake was recorded.
Experiment 3: effects of NP and LP diets on plasma amino acids.
To correlate levels of food intake with changes in circulating amino
acids, separate groups of rats maintained on a chow diet were
transitioned (during the mid-light cycle) to either NP or LP diets, with
individual groups (8/group) euthanized at day 0 (prior to dietary
treatment), or days 1, 2, or 4 after the dietary change. Animals were
killed in the fed state in the middle of the light cycle. Trunk blood was
collected at death, and plasma was isolated to assess circulating amino
acid levels.
Experiment 4: effects of NP and LP diets on brain amino acidbrain microdialysis. Brain microdialysis was performed to assess
LP-induced changes in brain amino acid concentrations. One week
following implantation of the microdialysis guide cannula into the
third ventricle, rats were placed into a sampling harness, and on the
following day, they were transitioned (during the mid-light cycle) to
the NP control diet. After 4 days of adaptation, half of the rats
remained on the NP diet, while the other half were offered LP diet for
2 days (4 or 5/group). On day 2, food was removed 3 h prior to lights
off, and the microdialysis probe was inserted. The CMA 11 microdialysis probes (CMA Microdialysis, Kista, Sweden; membrane length:
2 mm; membrane outer diameter: 0.24 mm) were flushed according to
the manufacturer’s recommendation prior to use and were inserted
during brief (⬃5 min) anesthesia on 3% isoflurane in oxygen. Perfusion was initiated as soon as the probe was implanted using CNS
perfusion fluid (M Dialysis AB, Stockholm, Sweden) (in mmol/l): 147
NaCl, 2.7 KCl, 1.2 CaCl2, 0.85 MgCl2, at a flow rate of 1 ␮l/min.
Perfusion began at food removal, and rats were given 3 h of recovery
prior to lights off. At lights off, sample collection began, with samples
collected every 30 min. One hour later (4 h of fasting), rats were
offered 3.2 g of their respective NP or LP diet, which was consumed
within 10 min in all animals. On a per gram basis, this test meal
represents 13.5% of 24-h intake at day 2 for NP and 10.1% of 24-h
intake for LP and was chosen to ensure that that all of the animals
consumed equal amounts of food within the specified 10-min window,
thus maintaining a similar energy intake but varying protein intake.
Sampling continued for another hour in this postprandial period, at
which point animals were killed, and trunk blood was collected to
assess plasma amino acid levels. In summary, microdialysis samples
were collected in 4-h fasted rats every 30 min for 1 h prior and 1 h
following a test meal. Daily food intake and body weight were
recorded throughout the 6-day protocol.
Experiment 5: effects of alterations in dietary amino acids on food
intake. To determine whether reduction in dietary leucine content is
necessary or sufficient to induce hyperphagia on the LP diet, rats were
exposed to one of five diets that altered dietary leucine content
independently from other amino acids or total protein (12/group;
starting body weight ⫽ 132 ⫾ 2 g). The five diets were 1) normalprotein control diet with 20% casein (NP); 2) low-protein diet with
10% casein (LP); 3) a modified NP diet in which half of the protein
was provided as casein and the other half was provided as free amino
acids (LP⫹All). Total protein and amino acid balance were identical
between the NP diet and the LP⫹All diet; 4) the LP⫹All diet with all
free amino acids removed except leucine (LP⫹LEU). Thus, this diet
was equivalent to the LP diet with the exception that leucine content
was equivalent to NP; and 5) the LP⫹All diet with the free leucine
removed (NP⫺LEU). Thus, this diet was equivalent to the NP and
LP⫹All diets, except leucine levels were reduced to those of the LP
diet. In summary, the LP⫹LEU diet and NP⫺LEU diets test whether
the reduction of dietary leucine to LP levels is necessary or sufficient
to induce hyperphagia. A precise description of these diets is included
in Table 1. Rats were initially placed on the NP diet for 3 days before
being randomly assigned to an experimental diet. Rats were maintained on these diets for 12 days, with food intake recorded daily.
Because rats seemed unresponsive to reductions in dietary leucine,
a very similar experimental protocol was used to test whether the
reduction of all three BCAAs (leucine, isoleucine, and valine) would
be sufficient to induce hyperphagia. A second group of rats (12/group;
starting body weight ⫽ 115 ⫾ 2 g) was placed on one of five
experimental diets consisting of the same control diets (NP, LP,
LP⫹All), as well as diets that manipulate all three BCAAs in a
manner similar to the changes in leucine above (LP⫹BCAA and
NP⫺BCAA). Diets are described in Table 1. As above, all rats were
initially placed on the NP diet for 3 days before being randomly
assigned to one of five treatments. Rats were maintained on these diets
for 7 days, with food intake recorded daily.
Experiment 6: effect of chronic intracerebroventricular amino acid
infusion on LP-induced hyperphagia. To assess whether elevations in
brain amino acids would be sufficient to block LP-induced hyperphagia, rats consuming the NP or LP diet were chronically infused with
saline or amino acids via an intracerebroventricular cannula. Indwelling lateral ventricular cannulas were implanted as described above.
After 7 days of recovery, rats were briefly anesthetized (3% isoflurane
in oxygen) to implant a subcutaneous osmotic minipump (Alzet
Model 2002; Durect, Cupertino, CA), which was attached to the
lateral ventricular cannula to allow direct brain infusion of amino
acids. All rats received 5 mg/kg body wt carprofen (Pfizer, New York,
NY) subcutaneously preoperatively as an analgesic. Minipumps contained either saline or increasing concentrations (5⫻, 10⫻, or 20⫻) of
a cell culture-based amino acid mixture (MEM amino acids cat. no.
25-030-CI; Mediatech, Manassas, VA). Contents and dose rate are
described in Table 2. Immediately following minipump surgery, rats
were assigned to either NP or LP diet, producing five groups:
NP⫹Saline, LP⫹Saline, LP⫹5⫻AA, LP⫹10⫻AA, and LP⫹20⫻AA
(8 –10/group). Daily food intake was recorded for 6 days.
Plasma and brain amino acid measures. Plasma amino acids were
measured using fluorometric HPLC via methods described previously
(28, 41). Separation of the o-phthaldialdehyde amino acid derivatives
was performed by gradient elution from a Supelcosil LC-18 column
(15 cm ⫻ 4.6 mm, 3 ␮m; Sigma, St. Louis, MO).
Statistical analysis. Data were analyzed using the SAS software
package (SAS V9; SAS Institute, Cary, NC) using a two-tailed t-test
or ANOVA using the general linear model procedure. When experiment-wide tests were significant, post hoc comparisons were made
using the LSMEANS statement with the PDIFF option, and represent
least significant differences tests for preplanned comparisons. All data
are expressed as means ⫾ SE, with a probability value of 0.05
considered statistically significant.
RESULTS
Low-protein diet induces hyperphagia. Consistent with previous experiments, rats consuming the LP diet exhibited a
marked and persistent increase in food intake relative to rats
Table 2. Amino acid mixture
Components
5⫻
10⫻
20⫻
L-Arginine·HCl
L-Cystine·2HCl
L-Histidine·HCl·H2O
L-Isoleucine
L-Leucine
L-Lysine·HCl
L-Methionine
L-Phenylalanine
L-Threonine
L-Tryptophan
L-Tyrosine·HCl
L-Valine
7.6
1.9
2.5
3.2
3.2
4.4
0.9
2.0
2.9
0.6
2.6
2.8
15.2
3.7
5.0
6.3
6.3
8.7
1.8
3.9
5.7
1.2
5.2
5.6
30.3
7.5
10.1
12.6
12.6
17.4
3.6
7.8
11.4
2.4
10.4
11.2
Dose rate per 24 h (in micrograms).
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BCAAS AND THE RESPONSE TO PROTEIN RESTRICTION
Food intake (g)
30
25
*
*
*
*
*
*
20
LP
15
NP
10
days on diet
Fig. 1. Low-protein diets increase food intake. Rats were transitioned from
chow diet to either an isocaloric low-protein (LP) or normal-protein (NP) diet,
and daily food intake was measured for 7 days. Starting on day 2, food intake
in the LP vs. NP groups significantly diverged, with intake remaining significantly different for the remainder of the study (*P ⬍ 0.01).
consuming control, NP diet. The increase in food intake was
first observed on day 2, and food intake remained elevated
throughout the 7-day experiment (P ⬍ 0.01; Fig. 1).
Leucine uniquely suppresses food intake among tested essential amino acids. Rats bearing third ventricular cannulas
were fasted for 24 h, and then injected with a single amino acid
(10 ␮g, 2 ␮l) or saline (n ⫽ 15–20/group). Tryptophan,
methionine, lysine, threonine (all essential), and serine (nonessential) all had no effect on food intake (P ⬎ 0.05), but
leucine produced a significant reduction in consumption of
standard chow following the 24-h fast (P ⫽ 0.017; Fig. 2A), as
shown previously (5, 23). Another group of rats were implanted with third ventricular cannulas, and following recovery, were placed on LP diet for 4 days. On day 4, rats were
injected with either leucine (10 ␮g, 2 ␮l) or saline (n ⫽
10/group) in the fed state, and 24-h food intake was recorded.
Leucine injection in the LP animals significantly decreased
food intake (P ⫽ 0.027; Fig. 2B). Taken together, these data
suggest that elevations in brain leucine are sufficient to reduce
food intake in rats consuming either chow or LP diet but that
this anorectic effect is not a common feature of all EAAs.
LP diet alters plasma amino acid concentrations. To determine whether changes in food intake in response to LP or NP
diets are mirrored by changes in circulating leucine or other
BCAAs, plasma amino acids were measured in rats consuming
either NP diet or LP diet for either 1, 2, or 4 days. Following
40
Food intake (g)
35
B
Control
AA treated
40
35
30
25
Food intake (g)
A
1 day of the LP diet, plasma BCAAs arginine, lysine, methionine, phenylalanine, threonine, tryptophan (all EAAs), and
asparagine were significantly decreased (P ⬍ 0.05; Table 3 and
Fig. 3). Plasma histidine (EAA), glycine, and ornithine showed
a tendency to decrease on LP at day 1 (0.05 ⬍ P ⬍ 0.1; Table
3). At day 2, plasma leucine was unaltered by the LP diet,
whereas arginine, isoleucine, threonine, tryptophan, valine (all
EAA), and taurine were significantly decreased (P ⬍ 0.05;
Table 3 and Fig. 3). Plasma lysine and methionine (both EAA)
showed a tendency to decrease on LP (P ⬍ 0.10), whereas
alanine and glutamine were significantly elevated on LP diet at
day 2 (P ⬍ 0.05; Table 3). At day 4, there was no difference
in BCAAs between NP vs. LP (Fig. 3), with only plasma
arginine, methionine, threonine, and tryptophan (all EAA)
significantly decreased on LP (P ⬍ 0.05), whereas taurine
showed a tendency to decrease (P ⬍ 0.10; Table 3). In contrast,
alanine, glutamine, glycine, histidine, ornithine, and serine
were significantly elevated on LP at day 4 (P ⬍ 0.05; Table 3),
whereas glutamic acid showed a tendency to increase (P ⬍
0.10; Table 3). These data demonstrate that reductions in
dietary protein lead to transient reductions in plasma BCAAs
that tend to normalize within a few days.
LP diets alter postprandial plasma and brain amino acids.
Brain levels of amino acids were measured on day 2 of LP
feeding, with third ventricular microdialysis samples collected
from 1 h before to 1 h after feeding of a controlled test meal.
Day 2 was selected on the basis of prior evidence showing that
food intake increases in LP animals on day 2. Rats used in the
current study reproduce this effect, as food intake was significantly increased in LP rats on day 2 prior to sample collection
(P ⬍ 0.05; Fig. 4A).
Preprandial brain BCAA levels were consistently lower in
LP vs. NP rats, but this difference failed to reach statistical
significance for any amino acid (P ⬎ 0.10, Table 4 and Fig. 4,
B and C). However, postprandial brain levels of all BCAAs,
alanine, asparagine, lysine, methionine, and threonine were
significantly lower in LP vs. NP rats (P ⬍ 0.05; Table 4 and
Fig. 4, B and C), whereas arginine, citrulline, and taurine
showed a tendency to decrease (P ⬍ 0.10; Table 4). Measurement of amino acids in blood plasma samples collected at the
end of the microdialysis experiment (1 h postprandial) revealed
a significant decrease of BCAAs in LP rats (P ⬍ 0.05; Fig.
4D), as well as reduced methionine, threonine, and tryptophan
(P ⬍ 0.05; Table 4). Only EAAs were reduced by LP, as
nonessential amino acid (NEAA) concentrations did not differ
between the NP and LP groups (Table 4 and Fig. 4D).
*
20
15
10
5
Control
Leucine
30
25
20
*
15
10
5
0
0
TRP
MET
LYS
THR
LEU
SER
LP
Fig. 2. Amino acids have variable effects on food
intake. A: rats bearing third ventricular cannula
were fasted for 24 h and subsequently injected
with a single amino acid (AA; 10 ␮g, 2 ␮l) ⬃2 h
prior to lights off, and 24-h food intake was
subsequently recorded. Amino acids tested were
tryptophan (TRP), methionine (MET), lysine
(LYS), threonine (THR), leucine (LEU), and serine (SER). Only leucine significantly suppressed
food intake (*P ⬍ 0.05). B: rats bearing third
ventricular cannula were placed on LP diet for 4
days. Rats were injected with leucine (10 ␮g, 2
␮l) ⬃2 h prior to lights off (rats were not fasted),
and 24-h food intake was recorded. Leucine significantly suppressed food intake in animals consuming the LP diet (*P ⬍ 0.027).
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BCAAS AND THE RESPONSE TO PROTEIN RESTRICTION
Table 3. Plasma amino acids in response to normal- and low-protein diets
0
1
2
4
Day/Diet
NP
NP
LP
NP
LP
NP
LP
Arginine
Histidine
Lysine
Methionine
Phenylalanine
Threonine
Tryptophan
Alanine
Asparagine
Aspartic acid
Citrulline
Glutamic acid
Glutamine
Glycine
Ornithine
Serine
Taurine
Tyrosine
109.3 ⫾ 11.5
69.3 ⫾ 5.4
593.8 ⫾ 82.8
71.9 ⫾ 5.3
70.4 ⫾ 3.1
393.1 ⫾ 34.9
84.9 ⫾ 6.1
572.5 ⫾ 30.2
87.2 ⫾ 5.5
47.7 ⫾ 9.2
82.8 ⫾ 5.4
144.2 ⫾ 12.8
787.7 ⫾ 41.1
179.1 ⫾ 17.7
73.0 ⫾ 9.1
170.7 ⫾ 13.8
94.4 ⫾ 12.1
150.1 ⫾ 9.5
116.7 ⫾ 4.2
81.5 ⫾ 3.7
720.3 ⫾ 21.0
76.3 ⫾ 4.3
72.6 ⫾ 2.5
453.1 ⫾ 17.7
96.4 ⫾ 4.2
617.4 ⫾ 35.3
99.4 ⫾ 7.0
32.4 ⫾ 2.3
76.0 ⫾ 3.6
125.5 ⫾ 5.5
781.6 ⫾ 38.5
142.7 ⫾ 7.0
59.0 ⫾ 3.2
161.2 ⫾ 9.1
98.4 ⫾ 5.7
175.4 ⫾ 11.4
85.9 ⫾ 1.8*
68.0 ⫾ 5.7#
557.0 ⫾ 44.8*
63.7 ⫾ 3.3*
62.7 ⫾ 2.0*
165.6 ⫾ 11.3*
55.5 ⫾ 2.8*
703.1 ⫾ 71.2
75.6 ⫾ 5.0*
32.2 ⫾ 2.0
70.8 ⫾ 2.4
126.6 ⫾ 5.4
830.1 ⫾ 47.7
110.3 ⫾ 14.4#
51.0 ⫾ 2.2#
150.3 ⫾ 8.6
89.6 ⫾ 6.6
147.1 ⫾ 12.6
124.9 ⫾ 5.5
74.2 ⫾ 2.4
680.9 ⫾ 43.7
68.4 ⫾ 2.7
64.5 ⫾ 3.4
416.8 ⫾ 33.4
86.0 ⫾ 6.9
551.2 ⫾ 31.1
88.0 ⫾ 4.4
33.2 ⫾ 2.7
86.7 ⫾ 4.1
125.3 ⫾ 6.4
778.4 ⫾ 33.4
148.2 ⫾ 3.8
55.8 ⫾ 3.8
164.9 ⫾ 8.1
112.3 ⫾ 8.7
139.0 ⫾ 10.9
91.7 ⫾ 5.0*
77.1 ⫾ 4.1
568.9 ⫾ 42.3#
60.2 ⫾ 3.7#
68.5 ⫾ 3.0
126.2 ⫾ 14.6*
62.1 ⫾ 4.8*
835.7 ⫾ 63.4*
86.4 ⫾ 2.9
32.3 ⫾ 1.5
83.5 ⫾ 4.2
142.4 ⫾ 7.7
921.2 ⫾ 26.4*
161.2 ⫾ 7.5
52.9 ⫾ 2.5
197.3 ⫾ 18.3
68.8 ⫾ 6.6*
133.7 ⫾ 12.4
126.7 ⫾ 6.9
73.8 ⫾ 3.7
669.6 ⫾ 43.6
72.3 ⫾ 4.5
70.4 ⫾ 4.6
463.0 ⫾ 39.2
97.5 ⫾ 8.7
548.4 ⫾ 36.2
100.3 ⫾ 7.6
33.5 ⫾ 1.4
132.3 ⫾ 44.6
124.9 ⫾ 9.5
849.2 ⫾ 49.4
147.7 ⫾ 9.4
53.2 ⫾ 2.0
194.7 ⫾ 12.0
121.0 ⫾ 14.2
148.8 ⫾ 9.7
100.5 ⫾ 3.4*
90.6 ⫾ 5.7*
627.9 ⫾ 57.6
61.0 ⫾ 2.7*
70.1 ⫾ 2.9
149.4 ⫾ 14.2*
64.9 ⫾ 4.1*
897.7 ⫾ 42.1*
107.5 ⫾ 2.2
43.7 ⫾ 6.4
93.9 ⫾ 2.6
158.2 ⫾ 13.3#
1055.1 ⫾ 53.0*
213.7 ⫾ 19.8*
71.2 ⫾ 6.0*
282.2 ⫾ 28.2*
83.3 ⫾ 12.8#
158.0 ⫾ 8.2
Values are expressed as means ⫾ SE. Rats remained on a NP diet or were transitioned to an isocaloric LP diet and were euthanized on days 0, 1, 2, and 4
following the transition. Plasma amino acids concentrations were measured via fluorometric HPLC. Values for plasma amino acids are given in ␮M. *P ⬍ 0.05
and #P ⬍ 0.10 compared NP vs. LP at each time point.
Leucine and BCAAs are neither necessary nor sufficient for
the detection of low dietary protein. While BCAAs fall in
response to the LP diet, this reduction appears to be only
transient compared with the persistent hyperphagia. Therefore,
we directly tested whether reductions in dietary leucine or
BCAA content were necessary or sufficient for the detection of
protein restriction. As expected, the LP diet induced a significant increase in food intake compared with the NP diet or the
LP⫹All diet (P ⫽ 0.001; Fig. 5). The LP⫹LEU diet induced
hyperphagia to a similar extent as the standard LP diet despite
containing normal levels of leucine. Similarly, food intake was
not increased in the NP⫺LEU group despite the reduction in
dietary leucine. These data suggest that the reduced leucine
content in the LP diet is neither necessary nor sufficient to
mediate hyperphagia on a LP diet.
To determine whether manipulation of all BCAAs would
more readily alter food intake, we repeated the above experimental design in a separate group of rats, except that all three
BCAAs were altered simultaneously. The results were similar.
As before, rats exhibited hyperphagia in the LP diets compared
with the normal protein diets (P ⬍ 0.01; Fig. 6), but reducing
dietary BCAA levels is neither necessary nor sufficient to
mediate hyperphagia on a LP diet.
Chronic central amino acid infusion does not blunt LPinduced hyperphagia. To assess whether elevations in brain
amino acids would be sufficient to block LP-induced hyperphagia, we chronically infused a mixture of amino acids into the
brain of rats consuming the LP diet. Saline-treated LP rats
consumed more food than saline-treated NP rats, as expected.
However, central amino acid infusions were not sufficient to
consistently suppress LP induced hyperphagia (Fig. 7).
DISCUSSION
A large number of studies indicate that variations in dietary
protein alter food intake and body weight (21). The majority of
these studies focus on the effect of high-protein diets to
suppress food intake, increase energy expenditure, and de-
crease adiposity (2, 11, 18). Far fewer experiments have
focused on the effects of low-protein diets, but the available
data suggest that low-protein diets increase food intake and
body adiposity (23, 38, 39). These data are consistent with the
concept of “protein leveraging”, which suggests that protein
intake is prioritized over energy (carbohydrate) intake (9, 16,
24, 31, 32). These data raise the obvious question of the
identity of the protein signal. How do animals detect dietary
protein content in general, and how do they avoid protein
deficiency, in particular? Circulating amino acids represent an
obvious candidate for this protein signal, and circulating
BCAAs (leucine, isoleucine, and valine) seem well suited to
act in the brain as signals of dietary protein intake. Leucine
suppresses food intake when given intracerebroventricularly,
directly impinges on hypothalamic neurons known to regulate
food intake (NPY/AgRP and POMC), and influences signaling
systems that are associated with feeding behavior (3, 5, 23, 29).
However, while early studies indicated that increasing dietary
BCAAs or leucine alone can reduce food intake (29), several
recent experiments have failed to demonstrate an effect of
leucine supplementation on food intake or hypothalamic signaling (15, 25, 45). In addition, the signaling systems thought
to mediate leucine action (mTOR, AMPK) are also influenced
by a wide variety of circulating and physiological signals
associated with energy, but not protein, metabolism (5, 20, 43).
Thus, the relevance of hypothalamic leucine signaling to a
specific regulation of protein intake remains unclear. Therefore, we chose to address the role of leucine in a different
context by focusing on the hyperphagia induced by a lowprotein diet, a setting in which animals are protein-restricted
but consume normal or elevated amounts of energy. The
primary hypothesis being tested was that reduced dietary protein/leucine intake leads to decreases in circulating and brain
leucine concentrations, with this fall of brain leucine providing
a signal of protein restriction that triggers hyperphagia.
To determine whether leucine is unique in its capacity to
regulate food intake, we first tested the relative efficacy of one
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BCAAS AND THE RESPONSE TO PROTEIN RESTRICTION
A
250
R315
NP
*
150
D
700
Total BCAAs (µM)
100
50
0
0
B
4
1
2
days on diet
Isoleucine (µM)
150
125
*
100
600
*
500
*
400
300
200
100
0
*
0
1
2
days on diet
4
E
75
2500
50
25
0
0
C
1
2
days on diet
4
350
Total EAAs (µM)
Leucine (µM)
LP
200
2000
*
1500
*
*
1000
Fig. 3. Plasma amino acids in rats acutely
placed on low-protein diets. Rats were transitioned from chow diet to either an isocaloric LP
or NP diet, with individual groups killed on day
0 (NP), or after 1, 2, or 4 days of being placed
on the low-protein and normal-protein diets.
Trunk blood was collected at death, and plasma
amino acids were measured via HPLC. Individual branched-chain amino acids (BCAAs)
(A: leucine, B: isoleucine, C: valine), total
BCAAs (D), and total essential amino acids
(EAAs; E) were affected by the LP diet (*P ⬍
0.05).
500
Valine (µM)
300
250
*
200
0
*
0
1
2
days on diet
4
150
100
50
0
0
1
2
days on diet
4
nonessential (serine) and five essential amino acids to suppress food intake following intracerebroventricular injection. Because testing all EAAs was beyond the scope of our
study, we chose amino acids representing an array of side
chains. For instance, lysine is charged, threonine and serine
are polar, and leucine, tryptophan, and methionine are
hydrophobic. Methionine is sulfur-containing. Tryptophan
is ringed and a precursor to serotonin. Serine, threonine, and
methionine are glucogenic, while leucine and lysine are
ketogenic, and tryptophan is both glucogenic and ketogenic.
Serine serves as a precursor for biosynthesis of various
protein and membrane lipid molecules and also interacts
with N-methyl-D-aspartate receptor. On the basis of these
observations, there is no obvious structural or metabolic
reason to explain why leucine was the only amino acid to
suppress food intake. However, a variety of studies in
peripheral tissues and cell culture demonstrate that leucine
is particularly effective at stimulating mTOR signaling (7, 8,
26, 30, 42). As such, we speculate that this signaling effect
of leucine underlies its unique effects on food intake.
However, because we only tested a subset of amino acids
and did so at only a single dose, it remains possible that an,
as yet, untested amino acid might also act in the brain to
suppress food intake (13), or that a tested amino acid might
work at a higher dose. Intracerebroventricular valine injec-
tion has been previously shown to have no effect on food
intake (3–5). However, it remains possible that isoleucine
would also exert effects on food intake, although isoleucine
is substantially less potent at stimulating mTOR activity
relative to leucine (30). Therefore, the primary conclusion is
not that leucine is the only amino acid capable of regulating
food intake following intracerebroventricular injection but
that the suppression of food intake is not common to all
EAAs. Importantly, the dose chosen (10 ␮g) would require
a CSF volume of 25 ml to produce the 3-␮M leucine
concentration observed via microdialysis. Considering that
the entire rat CSF volume is estimated to be around 0.1 ml,
the leucine injection is producing concentrations roughly
250-fold higher than normally observed within the ventricular space, at least acutely. Therefore, even if higher doses
of the tested amino acids were sufficient to suppress food
intake, it is unclear whether such an effect would have any
relevance to the physiological regulation of food intake.
We next tested whether fluctuations in circulating leucine
correlated with changes in food intake in response to both
normal- and low-protein diets. Our results show that plasma
BCAAs, as well as the majority of other EAAs, decrease within
1 day of LP diet exposure (Fig. 3, Table 3). For BCAAs, this
decrease is transient, however, with levels progressively increasing over time and normalizing relative to NP controls by
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BCAAS AND THE RESPONSE TO PROTEIN RESTRICTION
25
20
15
10
LP
5
NP
0
-2
-1
+1
+2
days relative to change of diet
3
*
2
1
E
200
*
*
*
100
50
40
*
NP-pre
LP-pre
NP-post
LP-post
30
20
*
10
0
LEU
Plasma amino acids (µM)
Plasma amino acids (µM)
*
4
C
0
D 250
150
*
5
Brain amino acids (µM)
30
B
*
*
Brain amino acids (µM)
Food intake (g)
A 35
ILE
VAL
Total BCAAs
Total EAAs
F
1600
NP
1400
LP
feeding
*
1200
0
1000
30
60
90
120 min
800
600
400
*
MS1
MS2
Pre
200
MS4
MS3
blood
Post
0
0
LEU
ILE
VAL
Total BCAAs Total EAAs
Fig. 4. Preprandial and postprandial brain and postprandial plasma amino acids in rats placed on a LP diet. Microdialysis in the third cerebral ventricle was performed
in rats consuming either NP or LP diet for 2 days. Microdialysis samples were collected at 30-min intervals from 1 h before to 1 h after feeding of a controlled test meal,
with amino acid concentrations averaged together for the two preprandial and two postprandial samples. One hour after the test meal, animals were killed, and trunk
blood was collected to assess plasma amino acid levels. Food intake (A) in LP and NP rats is shown prior to microdialysis. Brain concentrations of individual BCAAs
(B) and total EAAs (C) both before (pre) and following (post) a fixed NP or LP meal are shown. Plasma BCAAs (D) and total EAAs (E) were measured at the end of
the experiment. F: schematic drawing presents experimental design (MS, microdialysis sample). *P ⬍ 0.05. NP vs. LP at each time point.
day 4. In particular, plasma leucine levels were no longer
significantly different by day 2. These changes in circulating
BCAAs are consistent with the observed increases in food
intake, as the hyperphagia is first observed on day 2, and food
intake increases progressively through day 4. Interestingly,
several other EAAs failed to normalize by day 4, including
arginine, methionine, tryptophan, and, most notably, threonine.
Finally, the pattern for NEAAs is much different, in that
Table 4. Preprandial and postprandial brain and postprandial plasma amino acids in rats placed on a low-protein diet
Cerebrospinal Fluid
Plasma
Preprandial
Postprandial
Postprandial
Time/Diet
NP
LP
NP
LP
NP
LP
Arginine
Histidine
Lysine
Methionine
Phenylalanine
Threonine
Tryptophan
Alanine
Asparagine
Aspartic acid
Citrulline
Glutamic acid
Glutamine
Glycine
Ornithine
Serine
Taurine
Tyrosine
1.5 ⫾ 0.3
2.0 ⫾ 0.5
8.4 ⫾ 0.8
0.6 ⫾ 0.1
1.3 ⫾ 0.1
5.6 ⫾ 1.4
0.4 ⫾ 0.0
5.0 ⫾ 0.7
1.0 ⫾ 0.2
1.1 ⫾ 0.2
0.7 ⫾ 0.1
0.8 ⫾ 0.2
33.5 ⫾ 8.8
4.8 ⫾ 0.2
3.2 ⫾ 0.2
4.9 ⫾ 1.2
0.9 ⫾ 0.0
1.5 ⫾ 0.1
1.4 ⫾ 0.4
1.3 ⫾ 0.3
6.2 ⫾ 1.5
0.5 ⫾ 0.1
1.0 ⫾ 0.3
4.4 ⫾ 2.0
0.4 ⫾ 0.1
4.1 ⫾ 0.9
0.9 ⫾ 0.3
1.2 ⫾ 0.1
0.5 ⫾ 0.1
0.7 ⫾ 0.2
32.0 ⫾ 17.3
4.8 ⫾ 0.5
3.7 ⫾ 0.5
5.1 ⫾ 1.9
0.6 ⫾ 0.1
1.4 ⫾ 0.4
2.0 ⫾ 0.1
2.2 ⫾ 0.6
9.5 ⫾ 0.5
0.9 ⫾ 0.1
1.7 ⫾ 0.2
7.6 ⫾ 0.8
0.4 ⫾ 0.1
7.9 ⫾ 1.1
1.6 ⫾ 0.2
1.9 ⫾ 0.7
1.4 ⫾ 0.5
1.1 ⫾ 0.3
38.6 ⫾ 7.4
6.2 ⫾ 1.5
3.9 ⫾ 0.6
7.5 ⫾ 2.0
1.0 ⫾ 0.2
2.1 ⫾ 0.2
1.3 ⫾ 0.3#
1.2 ⫾ 0.3
6.2 ⫾ 1.0*
0.4 ⫾ 0.1*
1.2 ⫾ 0.2
4.0 ⫾ 1.2*
0.4 ⫾ 0.0
4.6 ⫾ 0.7*
0.9 ⫾ 0.2*
1.8 ⫾ 0.8
0.5 ⫾ 0.1#
0.7 ⫾ 0.1
28.5 ⫾ 12.6
4.9 ⫾ 0.5
3.5 ⫾ 0.6
5.3 ⫾ 1.4
0.5 ⫾ 0.1#
2.1 ⫾ 0.5
87.8 ⫾ 3.6
53.3 ⫾ 3.8
362.1 ⫾ 16.4
56.6 ⫾ 1.5
109.8 ⫾ 38.5
277.5 ⫾ 15.6
60.9 ⫾ 4.5
568.3 ⫾ 32.8
74.6 ⫾ 1.5
37.9 ⫾ 2.9
45.5 ⫾ 1.5
247.3 ⫾ 19.1
623.9 ⫾ 29.4
143.7 ⫾ 5.9
58.5 ⫾ 3.7
169.6 ⫾ 21.6
140.7 ⫾ 20.6
93.6 ⫾ 5.8
89.3 ⫾ 5.1
54.7 ⫾ 2.8
312.0 ⫾ 53.7
49.0 ⫾ 2.4*
94.6 ⫾ 17.4
182.5 ⫾ 21.9*
49.4 ⫾ 2.5*
642.8 ⫾ 34.5
64.4 ⫾ 4.6
36.7 ⫾ 4.3
43.5 ⫾ 1.1
208.2 ⫾ 49.1
581.9 ⫾ 27.3
127.2 ⫾ 10.7
51.3 ⫾ 2.2
151.9 ⫾ 10.7
121.8 ⫾ 15.6
85.7 ⫾ 2.9
Values are expressed as means ⫾ SE. Rats either remained on NP diet or were transitioned to an isocaloric LP diet. Two days following the transition,
cerebrospinal fluid (CSF) lysate samples were obtained with microdialysis before and after intake of an isocaloric NP or LP diet, respectively. Average of two
samples collected preprandial (⫺60 to ⫺30 min and ⫺30 to 0 min relative to food intake) and postprandial (0 to 30 min and 30 to 60 min after food intake)
are presented. One hour after the test meal animals were euthanized and trunk blood collected to assess plasma amino acid levels. Values for plasma amino acids
are given in ␮M. Amino acids concentrations were measured via fluorometric HPLC. *P ⬍ 0.05 and #P ⬍ 0.10 compared NP vs. LP at each time point.
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BCAAS AND THE RESPONSE TO PROTEIN RESTRICTION
30
25
20
NP
LP
LP+All
LP+LEU
NP-LEU
15
10
Avg daily intake (g)
Food intake (g)
30
*
*
25
20
15
10
-1
0
1
2
3
4 5 6 7
days on diet
8
9 10 11 12
NP
LP
LP+All
LP+LEU
NP-LEU
Fig. 5. Dietary leucine does not contribute to the hyperphagia induced by low-protein diets. Rats were placed on one of five isocaloric diets that varied in protein
and leucine content. Control diets included a NP, LP, or a modified NP diet, in which half of the protein was provided as casein and the other half was provided
as free amino acids (LP⫹All). The fourth diet (LP⫹LEU) consisted of the LP diet with leucine added back to equal the leucine content of the NP diet. The fifth
diet (NP⫺LEU) consisted of the LP⫹All diet minus leucine, such that leucine levels were equal to the LP diet. Left: daily food intake. Right: daily intake averaged
across days 3–12. Placing animals on LP induced a significant increase in food intake (*P ⬍ 0.05), regardless of leucine content.
NEAAs are generally increased by the LP diet (14). Taken
together, these data highlight the homeostatic regulation of
blood amino acid levels in the face of reduced protein intake.
LP diets induce relatively rapid decreases in liver and muscle
protein synthesis and amino acid metabolism, which together
serve to spare amino acids and buffer against dramatic decreases in their circulating levels (12, 33, 44). These data are
also consistent with the hypothesis that reductions in circulating amino acids may represent a trigger for hyperphagia,
although the rapid normalization of BCAAs levels despite
persistent hyperphagia makes their specific role in the maintenance of hyperphagia unclear.
Amino acids enter the brain through diverse transport mechanisms. While brain levels tend to correlate with circulating
levels, brain transporters are easily saturated, such that the
brain is buffered against marked changes in circulating amino
acids. Several amino acids also share a common transporter
and, thereby, compete for uptake into brain. To directly assess
changes in brain amino acid concentrations, microdialysis was
used within the third ventricle in both the preprandial and
postprandial state on day 2 of a LP diet, with day 2 chosen as
the first day of hyperphagia in our model. Interestingly, preprandial brain amino acid levels were consistently lower in LP
rats, but this difference was modest and did not reach statistical
significance for any amino acid. However, consumption of an
isocaloric NP or LP meal induced a clear postprandial difference in the CSF amino acids, primarily due to a postprandial
increase in the NP controls that failed to occur in the LP group.
Importantly, we observe clear differences in brain and blood
levels of BCAA concentrations between NP- and LP-fed rats in
the postprandial period, although it should be noted that plasma
and brain amino acids were not simultaneously measured over
time in the same animal.
Our data indicate that both circulating and brain concentrations of BCAAs are acutely reduced by the LP diet, particularly
in the postprandial period. However, this effect is transient, and
BCAAs levels in the blood normalized by day 4 despite
persistent increases in food intake beyond day 4. This rapid
normalization despite persistent hyperphagia raises considerable doubt regarding the role of BCAAs in the maintenance of
hyperphagia on the LP diet. To more directly test the role of
dietary leucine on LP-induced hyperphagia, isocaloric diets
30
25
20
NP
LP
LP+All
LP+BCAA
NP-BCAA
15
10
Avg daily intake (g)
Food intake (g)
30
*
25
*
20
15
10
1
3
4
5
days on diet
6
7
NP
LP
LP+All
LP+BCAA NP-BCAA
Fig. 6. Dietary BCAAs do not contribute to the hyperphagia induced by LP diets. Rats were placed on one of five isocaloric diets that varied in protein and BCAA
content, similar to above. Control diets included a NP, a LP, or a modified NP diet in which half of the protein was provided as casein and the other half was
provided as free amino acids (LP⫹All). The fourth diet (LP⫹BCAA) consisted of the LP diet with leucine, isoleucine, and valine added back to equal the BCAA
content of the NP diet. The fifth diet (NP-BCAA) consisted of the LP⫹All diet with leucine, isoleucine, and valine reduced to levels equal to that of the LP diet.
Left: daily food intake. Right: daily intake averaged across days 3–7. Placing animals on the LP diet induced a significant increase in food intake (*P ⬍ 0.05),
regardless of BCAA content.
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BCAAS AND THE RESPONSE TO PROTEIN RESTRICTION
NP+Saline
LP+Saline
LP+5XAA
LP+10XAA
LP+20XAA
25
30
Avg daily intake (g)
Food intake (g)
30
20
15
25
*
*
*
*
20
15
0
1
2
3
4
days on diet
5
6
NP+Saline LP+Saline LP+5XAA LP+10XAA LP+20XAA
Fig. 7. Brain infusion of amino acids did not block LP-induced hyperphagia. Rats bearing lateral ventricular cannula were continuously infused (via osmotic
minipump) with saline or increasing concentrations of a cell culture-based amino acid mixture. Coincident with initiation of intracerebroventricular infusion, rats
were also placed on either control (NP) or LP diet to determine whether brain amino acid infusion is sufficient to block LP-induced hyperphagia. LP⫹Saline
rats exhibited a significant increase in food intake relative to NP⫹Saline, beginning on day 2 of diet. Brain amino acid infusion failed to block this LP-induced
increase in food intake, except for a transient effect on day 2 with the highest dose (20⫻AA). Left: daily food intake. Right: average intake from days 2– 6.
*P ⬍ 0.05 vs. NP⫹Saline.
were used in which leucine was manipulated separately from
total protein. Because our diets are 20% and 10% casein,
respectively, we manipulated leucine levels within this dietary
range, and, as such, tested whether reduced dietary leucine
alone was sufficient to trigger hyperphagia, or if maintaining
normal levels of dietary leucine was sufficient to block hyperphagia. Unexpectedly, the rats were unresponsive to the
reduction of leucine, suggesting that reductions in dietary
leucine alone are neither necessary nor sufficient to signal
reduced dietary protein content. To test whether the combination of all BCAAs might alter food intake, the same experiment
was repeated with the exception that all three BCAAs were
manipulated. The result was the same, as the reduction of
dietary BCAAs failed to trigger hyperphagia, and the maintenance of control levels failed to block hyperphagia. Importantly, rats on the control and the LP⫹All diet consumed very
similar amounts of food in both studies, indicating that the
inclusion of free amino acids in the diet did not markedly alter
feeding behavior. Taken together, these data do not support the
hypothesis that reduction in either leucine alone or the combination of all BCAAs represents a unique signal that triggers
LP-induced hyperphagia. Importantly, our results are supported by previous work demonstrating that supplementing a
LP diet with BCAAs had no effect on protein selection (1), and
more recent work indicating that administration of leucine via
drinking water, gavage, gastric infusion, or intraperitoneal
injection all failed to suppress food intake (13, 15, 25, 45).
In light of these negative findings, it is tempting to speculate
as to the composition of amino acids needed to trigger hyperphagia. One assumes that food intake would eventually
increase if we continued to remove amino acids from the
LP⫹All diet. Since plasma arginine, threonine, tryptophan, and
methionine were all reduced in LP-fed animals on day 4 (Table
3), it seems possible that reduced dietary intake of these amino
acids may collectively contribute to the LP-induced hyperphagia. However, it remains unclear whether the detection involves direct amino acid signaling within the brain, or alternatively whether the restriction of amino acids is detected
elsewhere (gut, liver, muscle) and then secondarily signaled to
the brain. We tested this question by determining whether
direct brain infusion of a mixture of EAAs is sufficient to block
LP-hyperphagia. While the highest dose tended to reduce food
intake on the second and third day of infusion, this effect
disappeared rapidly, and food intake was no different from
saline-infused LP rats in any of the EAA-infused groups on
days 4 – 6. As such, these data fail to support the hypothesis
that increases in brain amino acids are sufficient to block
LP-induced hyperphagia.
The work described here focused specifically on the hypothesis that alterations in brain leucine action might contribute to
the hyperphagia induced by a LP diet. This hypothesis was
based, in part, on our work and the work of others demonstrating that leucine suppresses food intake following intracerebroventricular injection, in part, on the evidence that leucine is
particularly potent in activating intracellular signaling, and, in
part, on the evidence that the brain is critically involved in the
response to the dietary depletion of a single EAA. In this latter
response, deprivation of a single dietary EAA activates the
anterior piriform cortex (APC), specifically via reduction in the
concentration of the limiting EAA in the APC (10). It should
be mentioned, however, that this APC-dependent recognition
of EAA deficiency produces a rejection (decreased intake) of a
novel EAA-deficient meal, whereas in our work, the general
restriction of dietary protein produces hyperphagia.
There is a variety of sites and mechanisms through which
dietary amino acids could alter food intake, in particular,
within the gastrointestinal (GI) tract, portal vein, or liver.
Recent work demonstrates that intragastric infusion of glutamate, arginine, or lysine is sufficient to reduce food intake and
that the effect of lysine requires an intact vagus nerve, while
the effect of arginine and glutamate requires the brain stem
area postrema (13). In this experiment, the amino acids are
individually infused in excess as opposed to being removed
from a mixed diet as in our current work, but it is also
interesting to note that intragastric leucine was shown to have
no effect. Amino acids in the intestine are well known to
stimulate the release of anorexigenic gut peptides, such as
cholecystokinin, glucagon-like peptide-1, and peptide YY, and
it seems likely that the action of these hormones on vagal
afferents or directly in the brain contributes to the anorectic
effect of protein (6). However, these gut hormones are also
induced by other macronutrients, suggesting that they are
AJP-Regul Integr Comp Physiol • doi:10.1152/ajpregu.00116.2014 • www.ajpregu.org
BCAAS AND THE RESPONSE TO PROTEIN RESTRICTION
unlikely to represent unique signals of protein restriction.
Amino acid sensors also exist within the gut and the hepatoportal circulation, leading to the activation of the vagus nerve
(34 –36), although hepatic vagal afferents do not seem essential
to mediate the anorectic effects of a HP diet (17). Protein
restriction also induces significant changes in hepatic metabolism (14), and thus, amino acid signaling within the liver may
also contribute to the detection of protein restriction. Despite
the evidence that amino acids impact hepatic function, gut
hormone secretion, and vagal activity, the specific role of these
mechanisms in mediating the response to dietary protein,
particularly protein restriction, remain unclear (21).
Perspectives and Significance
Acute brain administration of leucine is sufficient to reduce
food intake, and blood and brain levels of leucine acutely fall
coincident with the initiation of hyperphagia in animals on LP
diets. While these data suggest that leucine is positioned to
regulate food intake during dietary protein restriction, reductions in dietary leucine or BCAA content had no effect on food
intake, and chronic central amino acid infusion was not sufficient to block LP-induced hyperphagia. Taken together, we
find no evidence that variations in dietary or brain BCAAs
uniquely contribute to the hyperphagia induced by dietary
protein restriction. These results do not indicate that BCAAs
are unimportant. Instead, they suggest that the physiological
mechanisms that trigger hyperphagia on a LP diet require
reductions in additional amino acids beyond just the three
BCAAs, and that this hyperphagia is not likely dependent on
reductions in brain amino acid content.
ACKNOWLEDGMENTS
The authors would like to thank Dr. Michael Pellizon of Research Diets for
assistance in diet formulation. The authors thank the staff of the PBRC
Comparative Biology Core for their skillful assistance and excellent technical
support. This project/work used facilities within the Animal Metabolism &
Behavior Core, Genomics Core, and/or Cell Biology and Bioimaging Core at the
Pennington Biomedical Research Center, which are supported, in part, by Centers
of Biomedical Research Excellence (P20GM103528) and Nutrition Obesity Research Centers (P30DK072476) grants from the National Institutes of Health.
Present address: T. M. Henagan, Department of Nutrition Science, Purdue
University, West Lafayette, IN 47907.
GRANTS
This work was supported by a National Institutes of Health grant to C. D.
Morrison (5R01DK081563). T. Laeger was supported by a research fellowship
from the Deutsche Forschungsgemeinschaft LA 3042/2-1.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the authors.
AUTHOR CONTRIBUTIONS
Author contributions: T.L., T.M.H., H.M., R.J.M., S.M.H., and C.D.M.
conception and design of research; T.L., S.D.R., T.M.H., D.H.F., M.T., A.A.,
and C.D.M. performed experiments; T.L., S.D.R., T.M.H., D.H.F., M.T., A.A.,
S.M.H., and C.D.M. analyzed data; T.L., H.M., R.J.M., S.M.H., and C.D.M.
interpreted results of experiments; T.L. and C.D.M. prepared figures; T.L.,
S.D.R., and C.D.M. drafted manuscript; T.L., S.D.R., T.M.H., H.M., S.M.H.,
and C.D.M. edited and revised manuscript; T.L., S.D.R., T.M.H., D.H.F.,
M.T., A.A., H.M., R.J.M., S.M.H., and C.D.M. approved final version of
manuscript.
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