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R E V I E W Open Access
Novel metabolic and physiological
functions of branched chain amino acids: a
review
Shihai Zhang
1,2
, Xiangfang Zeng
1*
, Man Ren
3
, Xiangbing Mao
4
and Shiyan Qiao
1
Abstract
It is widely known that branched chain amino acids (BCAA) are not only elementary components for building
muscle tissue but also participate in increasing protein synthesis in animals and humans. BCAA (isoleucine, leucine
and valine) regulate many key signaling pathways, the most classic of which is the activation of the mTOR signaling
pathway. This signaling pathway connects many diverse physiological and metabolic roles. Recent years have
witnessed many striking developments in determining the novel functions of BCAA including: (1) Insufficient or
excessive levels of BCAA in the diet enhances lipolysis. (2) BCAA, especially isoleucine, play a major role in
enhancing glucose consumption and utilization by up-regulating intestinal and muscular glucose transporters. (3)
Supplementation of leucine in the diet enhances meat quality in finishing pigs. (4) BCAA are beneficial for
mammary health, milk quality and embryo growth. (5) BCAA enhance intestinal development, intestinal amino acid
transportation and mucin production. (6) BCAA participate in up-regulating innate and adaptive immune responses.
In addition, abnormally elevated BCAA levels in the blood (decreased BCAA catabolism) are a good biomarker for
the early detection of obesity, diabetes and other metabolic diseases. This review will provide some insights into
these novel metabolic and physiological functions of BCAA.
Keywords: Amino acid transporters, Glucose transporters, Gut health, Immunity, Lipolysis, Mammary health, Meat
quality, Milk production
Background
The branched chain amino acids (BCAA: leucine, isoleu-
cine, and valine) are essential amino acids and must be
obtained from the diet. BCAA not only act as building
blocks for tissue protein (accounting for 35% of the
essential amino acids in muscle) [1], but also have other
metabolic functions [1]. Among the three BCAA, leucine
earns the greatest reputation for its specific function in
activation of the mTOR signaling pathway. Since the
1970s, the role of leucine in enhancing protein synthesis
has been reported both in vitro and in vivo [2, 3]. More
recently, BCAA have been extensively used as
performance-enhancing supplements for body builders
and fitness enthusiasts [4, 5]. Besides playing a vital role
in protein metabolism, a variety of physiological and
metabolic functions have been reported for BCAA. For
instance, BCAA were reported to increase the secretion
of insulin [6]. However, increased level of plasma BCAA
have also been reported to lead to insulin resistance or
type 2 diabetes mellitus. One possible mechanism for
this is that persistent activation of mTOR signaling path-
way uncouples the insulin receptor from insulin receptor
substrate 1 [7]. Another possible mechanism is accumu-
lation of toxic BCAA metabolites (caused by abnormal
BCAA metabolism) may trigger mitochondrial dysfunc-
tion which is associated with insulin resistance [8].
More recently, BCAA have been reported to partici-
pate in lipolysis, lipogenesis, glucose metabolism, glu-
cose transportation, intestinal barrier function and
absorption, milk quality, mammary health, embryo
development, and immunity [9–14]. In addition, levels
of BCAA in the body can act as a biomarker for the
early detection of chronic diseases in humans [15]. The
* Correspondence: zengxf@cau.edu.cn
1
State Key Laboratory of Animal Nutrition, College of Animal Science and
Technology, China Agricultural University, No.2 Yuanmingyuan West Road,
Haidian District, Beijing 100193, People’s Republic of China
Full list of author information is available at the end of the article
© The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Zhang et al. Journal of Animal Science and Biotechnology (2017) 8:10
DOI 10.1186/s40104-016-0139-z
main objective of this review is to provide insights into
new developments in BCAA research as well as their
implications for both animal husbandry and human
health.
Metabolism of BCAA
The metabolism of BCAA is well established. However,
this should be addressed before we start looking into the
detail functions of BCAA as this can provide the reader
with a better understanding of this paper. BCAA are not
degraded directly in the liver and most of them are avail-
able for metabolism in skeletal muscle and other tissues.
However, the liver can oxidize BCAA after they are con-
verted into α-ketoacids in other tissues [16]. The main
steps of BCAA catabolism are listed below (Fig. 1).
Firstly, with the participation of branched-chain amino-
transferase (BCAT), BCAA are converted into branched-
chain α-ketoacids (leucine to α-ketoisocaproate, valine
to α-ketoisovalerate, and isoleucine to α-keto-β-methyl-
valerate) by removing their amino group. Subsequently,
branched-chain α-ketoacids are decarboxylated by
branched-chain α-ketoacid dehydrogenase (BCKD). Fi-
nally, these BCAA metabolites are catabolized by a series
of enzyme reactions to final-products (acetyl-CoA from
leucine, succinyl-CoA from valine, and both acetyl-CoA
and succinyl-CoA from isoleucine), which enter the
TCA cycle.
BCAA and fatty acid metabolism
In humans, consumption of diets with an increased pro-
tein and reduced carbohydrate content enhances weight
loss with greater loss of body fat and less loss of lean
body mass [17, 18]. In recent years, BCAA have been
considered as novel therapeutic tools for controlling
obesity and its related metabolic disorders, such as dia-
betes and insulin resistance, by enhancing exercise per-
formance, regulating the composition of body protein
and its properties, and controlling glucose tolerance,
which are all related to improved health and fitness [16].
Studies in mice have indicated that diet-induced obes-
ity mice in an isoleucine treatment (final concentration
of 2.5% isoleucine in drinking water) had almost 6%
lower body weight gain and 49% less epididymal white
adipose tissue mass compared with the control treat-
ment, with higher levels of hepatic protein CD36/fatty
acid translocase, PPARα, and uncoupling protein (UCP)
2 and muscular levels of UCP3 [13]. Similarly, when
dietary energy was restricted, leucine supplementation
was found to increase fat loss and enhance muscle
protein synthesis [19].
Interestingly, several studies have reported that supply-
ing animals with a BCAA deficient diet increased
lipolysis. Studies in mice discovered that feeding a
leucine-deficient diet for 7 d suppressed lipogenesis in
the liver [20] and also increased fat lipolysis in white adi-
pose tissue [21]. Furthermore, isoleucine or valine
deprivation also induced fat mass loss in mice [22]. Simi-
larly, in female broiler chickens, low dietary BCAA levels
reduced fatty acid synthesis and enhanced fatty acid-
oxidation by up-regulating hepatic lipogenic gene ACCα
and SCD-1 expression (ACCαis the enzyme for carb-
oxylation of acetyl-CoA to malonyl-CoA which is the
Fig. 1 Pathway of branched chain amino acid catabolism. BCAA are catabolized to acetyl-CoA and/or succinate-CoA and subsequently enter the
TCA cycle. The main steps of the catabolic reactions (transamination by BCAT and decarboxylation by BCKD) are shown. With the help of BCAT,
BCAA are catabolized into branched-chain α-ketoacids which are subsequently decarboxylated by BCKD. Finally, all the BCAA metabolites are
catabolized by a series of enzyme reactions to final products and enter the TCA cycle
Zhang et al. Journal of Animal Science and Biotechnology (2017) 8:10 Page 2 of 12
rate-limiting step for both synthesis and elongation of
fatty acid synthesis while SCD-1 catalyzes the biosyn-
thesis of monounsaturated fatty acids from dietary
lipids) without affecting growth performance, and is
likely mediated through the AMPK-mTOR-FoxO1 path-
way [23].
A possible mechanism of a BCAA deficient diet in
enhancing lipolysis is the activation of the GCN2 path-
way. It was demonstrated that a leucine deficient diet
resulted in reduction of food intake and weight lost in
both GCN2
+/+
and GCN2
−/−
mice, but only resulted in
loss of liver mass and abdominal adipose mass in GCN2
+/+
mice [20].
Taken together, both insufficient or excessive levels of
BCAA in the diet could be detrimental to lipid metabol-
ism. Supplementation of BCAA increased acetyl CoA
levels in cells which subsequently inhibited the activity
of pyruvate dehydrogenase. The preference for the cellu-
lar energy source was shifted from carbohydrate to lipid.
Also, BCAA up-regulates hepatic fatty acid translocation
and fatty acid oxidation gene expression [13]. Compared
with BCAA supplementation of the diet, feeding animals
with a BCAA deficient diet dramatically reduces food in-
take by activating the GCN2 signaling pathway, which
might participate in lipolysis (down-regulating lipogen-
esis genes or up-regulating lipolysis genes) in the liver
and adipose tissue. At present, studies focusing on fatty
acid metabolism are limited. Although some contradic-
tions exist among different studies, the intimate relation-
ship between BCAA metabolism and fatty acid
metabolism cannot be denied and more research is
needed in the future to explore these relationships.
BCAA and glucose transportation
Accumulating evidence indicates a strong connection
between amino acids and plasma glucose levels [24].
Branched chain amino acids have been demonstrated to
strongly enhance glucose consumption and utilization
[14]. In an animal oral glucose tolerance test, both iso-
leucine and leucine prevented a rise in plasma glucose
concentrations, and the effect of isoleucine was greater
than the other BCAA [14]. In a C2C12 myotubes ex-
periment, both leucine and isoleucine stimulated glu-
cose uptake [14]. Nishitani et al.[25] and Doi et al.
[26] observed similar results showing that isoleucine
participated in plasma glucose uptake in the rat. A
hypothesis for the mechanism through which isoleu-
cine and leucine regulate the serum glucose levels
might be due to an increase in muscle glucose
uptake, whole body glucose oxidation and a decrease
in hepatic gluconeogenesis [27].
The fact that BCAA enhance glucose uptake with acti-
vation or up-regulation of glucose transporters has been
widely demonstrated [14, 25]. Leucine increases glucose
uptake by up-regulating the translocation of GLUT4 and
GLUT1 in rat muscle [25]. Similarly, another experiment
reported that leucine enhances the expression of GLUT4
glucose transporter and 2-deoxyglucose uptake in
C2C12 cells [14]. Scientists suggest two hypotheses to
interpret the mechanism through which leucine regu-
lates muscular glucose transporters. Firstly, leucine
enhances translocation of GLUT1 and GLUT4 by up-
regulating insulin levels [28–30]. Secondly, leucine
increases glucose uptake in skeletal muscle via the PI3K
and PKC signaling pathways [14] both of which are
associated with GLUT4 translocation [31].
Compared with leucine, research focusing on the
mechanism through which isoleucine acts is limited. Re-
cent studies done in our lab demonstrate that feeding
weanling pigs an isoleucine deficient diet down-regulates
the protein expression of GLUT1 in red muscle and
GLUT4 in red muscle, white muscle and intermediate
muscle (Fig. 2) [32]. Furthermore, our experiments
showed that an isoleucine deficient diet suppresses the
expression of intestinal glucose transporter SGLT-1 in
the duodenum, jejunum and ileum and GLUT2 in the
duodenum and jejunum (Fig. 2). The function of isoleu-
cine in enhancing glucose uptake and muscular glucose
transporter expression (GLUT1 and GLUT4) was also
demonstrated in C2C12 myotubes in our study. How-
ever, the underlying mechanisms through which it
functions are still unknown.
Collectively, BCAA regulate the expression and trans-
location of muscular or intestinal glucose transporters
through insulin-dependent or insulin-independent ways.
These findings have important implications in that
BCAA could enhance muscle growth and intestinal de-
velopment by increasing the local glucose uptake for ani-
mals and humans.
BCAA and protein synthesis
Since 1999, Joshua C. Anthony, the pioneer in leucine
functional research, conducted a series of experiments
regarding the effects of leucine on muscle protein syn-
thesis and its underlying mechanisms (Fig. 3). Firstly, his
team observed that leucine stimulates the recovery of
skeletal muscle protein synthesis after exercise, inde-
pendent of increased plasma insulin [33]. Their studies
also revealed that leucine enhances muscle protein syn-
thesis via the mammalian target of rapamycin (mTOR)
pathway leading to phosphorylation of its downstream
target proteins, eukaryotic initiation factor 4E-binding
protein (4E-BP1) and p70 ribosomal S6 kinase 1 (S6K1)
[34, 35]. Since then, many experiments have been con-
ducted which strongly support their results [5, 36]. Leu-
cine has been shown to stimulate muscle protein
synthesis in rats [19, 37, 38], pigs [39–41] and humans [5,
42, 43]. The team of Teresa A. Davis evaluated the
Zhang et al. Journal of Animal Science and Biotechnology (2017) 8:10 Page 3 of 12
function of leucine in neonates. They found leucine
has unique anabolic properties and the supplementation
of leucine or its metabolites α-ketoisocaproic acid and
β-hydroxy-β-methylbutyrate strongly increase muscle
protein synthesis in neonates [44–46]. Supplementation of
leucine in a protein deficient diet had a strong positive
connection to protein synthesis [47]. Interestingly, some
studies reported that supplementation of leucine in a
chronically restricted protein and energy diet only
enhanced mTOR pathway activation without increasing
Fig. 2 Isoleucine up-regulates intestinal and muscular transporters. GLUT1 and GLUT4 are vital glucose transporters in muscle. SGLT1 and GLUT2
are important glucose transporters in the small intestine. Isoleucine could potentially increase muscle growth and intestinal development and
health by up-regulating the protein expression of GLUT1 and GLUT4 in muscle and enhancing the expression of SGLT1 and GLUT2 in the
small intestine
Fig. 3 Leucine increases protein synthesis by activation of the mTOR signaling pathway. Leucine enhanced muscle synthesis via the mammalian
target of rapamycin (mTOR) pathway leading to phosphorylation of its downstream target proteins, eukaryotic initiation factor 4E-binding protein
(4E-BP1) and p70 ribosomal S6 kinase 1 (S6K1). Under unphosphorylated conditions, 4EBP1 tightly binds to eIF4E, forming the inactive eIF4E ·
4EBP1 complex. During anabolic conditions, mTORC1 induces the phosphorylation of 4EBP1, resulting in the dissociation of eIF4E from the
inactive complex and allowing eIF4E to form an active complex with eIF4G. The process of association of eIF4E with eIF4G is obligatory for the
binding of the 43S pre-initiation complex with mRNA. S6K1 is another mTORC1 substrate that participates in the regulation of mRNA translation.
This kinase plays an important role in the regulation of terminal oligopyrimidine mRNA which is responsible for the translation of proteins
involved in the protein synthetic apparatus
Zhang et al. Journal of Animal Science and Biotechnology (2017) 8:10 Page 4 of 12
muscle protein synthesis in neonatal pigs [48]. This
indicates that the stimulation of protein synthesis by
leucine is dependent on the availability of other
amino acids [49].
Recently, the synergistic effect between leucine and lep-
tin, and the effect of leucine on meat quality has been re-
ported by our lab. In our study, we found that leucine
stimulated the expression of leptin and its muscular re-
ceptor [50]. In addition, the combination of leptin and leu-
cine synergistically regulated protein metabolism in
skeletal muscle both in vitro and in vivo [51]. In an experi-
ment with finishing pigs, we found that supplementation
with leucine (1.25%) could enhance pork texture by en-
hancing protein deposition and improving meat quality
[52]. Similarly, another experiment in finishing pigs re-
ported that leucine addition increased juiciness accom-
panying the increase in intramuscular fat content
occurring with a protein deficient diet [53]. However, sup-
plementation of excess leucine (3.75%) in the diet changes
plasma amino acid-derived metabolites, which may limit
the use of high Leu diets to treat muscle atrophy [54].
Therefore, a high dose of leucine could be toxic and find-
ing a suitable supplementation level of leucine is vital for
future use.
In conclusion, the effect of leucine in increasing pro-
tein synthesis via the mTOR signaling pathway is widely
known, the function of which might be enhanced by
leptin. A reasonable supplementation level for leucine
could improve meat quality.
BCAA and feed intake
The function of central leucine infusion on feed intake
inhibition has been demonstrated in many studies [55, 56].
The mTOR signaling pathway plays a vital role in the
brain to detect nutrient availability and regulate en-
ergy balance [57]. In an experiment with rats, Cota et
al.[57] demonstrated that mTOR signaling is con-
trolledbyenergystatusinspecificregionsofthe
hypothalamus and colocalizes with neuropeptide Y
and proopiomelanocortin neurons in the arcuate nu-
cleus. However, the function of leucine on feed intake
is different when leucine is supplied in the diet. Many
experiments demonstrated that extra supplementation
of leucine in the diet not increase feed consumption
in animals [58–61]. The divergent results caused by
oral or central leucine supplementation might be ex-
plained by the capacity of leucine to cross the blood–
brain barrier and reach the central neural system.
Although extra supplementation of BCAA in the diet
does not further increase the feed intake, the function of a
BCAA-deficient diet in down-regulating feed intake can
not be ignored. Gloaguen et al.[62] conducted a trial to
test if feed intake was affected after ingestion of Val- and
Val + diets with an excess of Leu. They found that prior
ingestion of the Val- test diet resulted in a 14% reduction
in feed intake compared with Val + test meal. Zhang et al.
[63] reported that feeding piglets with 17% crude protein
BCAA-deficient diet significantly decreased feed intake by
42%. As BCAA are essential amino acids for animals, the
reduction of feed intake could be interpreted as a function
of the unbalance essential amino acid levels in the serum,
which are regulated with the activation of GCN2 signaling
pathway [64].
BCAA and mammary function
A significantly increased whole-body BCAA catabolism
has been observed during lactation compared with non-
lactating counterparts [65, 66]. BCAA catabolism could
enhance the syntheses of glutamate, glutamine, aspartate,
alanine, and asparagine in the mammary gland and in-
crease the production of milk for suckling neonates
[11, 12]. In addition, the major leucine transporter LAT1
is a limiting factor for the synthesis of glutamate and aspar-
tate in mammary tissue [67]. Concurrently, the activity of
BCAT and BCKD (two vital enzymes in BCAA catabolism)
are increased in mammary tissue during lactation [68],
which might be caused by reductions in insulin and growth
hormone or increases in cortisol and glucagon [69].
Accumulating evidence indicates the obvious connec-
tion among BCAA, milk production and neonatal piglet
performance. An experiment in sows demonstrated that
supplementation of BCAA in the diet (Control group:
16% CP vs. Treatment group: 23% CP + BCAA) in-
creased milk protein secretion but not milk yield [70]. In
early lactation in fistulated dairy cows, compared with
abomasally infused EAA ones, omission of leucine or
BCAA decreased protein yield about 12% and 21%, re-
spectively [71]. A recent study found that increasing the
total dietary Val:Lys ratio from 0.84:1 to 0.99:1 increased
milk concentrations of isoleucine and valine [72]. These
results indicate that the level of BCAA (especially leu-
cine) in the diet plays a vital role in determining milk
protein percentage. The underlying mechanism for a de-
ficiency of BCAA impairing milk protein production is
due to the deactivation of mTORC1-mediated up-
regulation of eIF2Bεand eIF2αabundance [73]. How-
ever, another 2 × 2 × 2 factorial study in sows (two levels
of valine (0.80 and 1.20%), isoleucine (0.68 and 1.08%),
and leucine (1.57 and 1.97%)) found that only supple-
mentation of valine tended to increase milk nitrogen,
but not isoleucine or leucine [74]. For neonatal piglets,
this research found that increasing dietary valine level
from 0.8% to 1.2% in sows is vital for increasing litter
weaning weight [74].
The function of BCAA on milk production might be
manipulated via the mammary cells (Fig. 4). In mammary
epithelial cells, BCAA could stimulate their growth and
proliferation, enhance their functional differentiation and
Zhang et al. Journal of Animal Science and Biotechnology (2017) 8:10 Page 5 of 12
increase their longevity [75]. Both leucine and isoleucine
have been shown to enhance the fractional protein synthe-
sis rates in bovine mammary cells with the phosphor-
ylation of mTOR and rpS6, or mTOR, S6K1, and
rpS6 respectively [76], while decreasing the abundance
of proteasome protein, ubiquitinated protein, and the rate
of protein degradation [77]. The mechanism through
which valine functions is still unknown.
These novel findings not only advance our under-
standing of BCAA regulation of lactation but also
provide a new strategy to improve milk production by
livestock and humans.
BCAA, blastocyst development and fetal growth
Early embryo growth and development of the fetus de-
pend entirely on maternal nutrition. Alterations in fetal
health are strongly associated with the development of
chronic diseases later in adult life [78]. BCAA have been
implicated as one of the vital elements in fetal develop-
ment. Compared with women with healthy fetuses, preg-
nant women with fetuses with intrauterine growth
retardation (IUGR) have lower plasma concentrations of
BCAA in the umbilical artery and vein [79]. Protein syn-
thesis is important for early embryo development.
Among all the BCAA, leucine is the most important as
it stimulates protein synthesis in skeletal muscle and
other tissues through the mTOR signaling pathway [80].
Supplementation of BCAA (1.8% L-Leu, 1.2% L-Val, and
1% L-Ile) relieves IUGR syndrome induced by a low-
protein maternal diet through activation of the mTOR
signaling pathway [10]. Furthermore, a BCAA-
supplemented diet is reported to improve the gene and
protein expression of IGF-1 and IGF-2 in fetal liver
which could ameliorate fetal growth restrictions [79].
However, another study found L-leucine (at doses of 300
or 1,000 mg/kg body weight) administered orally during
organogenesis did not affect the outcome of pregnancy.
in rats [81]. The conflict in results might be caused by
different dietary leucine concentrations and BCAA
combinations. It is apparent that leucine up-regulates
fetal growth by the enhancement of protein synthesis
and secretion of hormones.
Besides being necessary for the development of the
embryo, amino acids enhance embryo implantation by
improving blastocyst quality. Researchers have found
that amino acids are necessary for cultivating mouse
embryos in vitro [82] which might be because of the
embryonic requirement for AA as basic nutrients. Sub-
sequently, amino acids are proved to induce trophecto-
derm motility and mouse embryo implantation via
activation of the mTOR signaling pathway [83–85]. As
one of the most vital roles for leucine is activating the
mTOR signaling pathway, this indicates that leucine
might participate in blastocyst development. Recently, it
has been reported that up-regulation of leucine trans-
porter SLC6A14 induces blastocyst activation [86, 87].
All this evidence indicates that leucine has an important
function in blastocyst development. However, the opti-
mal levels of BCAA required during pregnancy and
lactation for animals and humans are still unknown.
BCAA and gut function
In addition to glutamine and asparagine, large amounts
of BCAA are consumed and oxidized in the intestine.
BCAA were catabolized in jejunal mucosal cells with a
high activity of cytosolic BCAT and about 30% of the
BCAA-derived branched-chain α-ketoacid were decar-
boxylated by BCKD [88, 89]. Several studies have found
that BCAA participates in intestinal amino acid trans-
porter expression [90]. Supplementing 1.4 g L-leucine/
Fig. 4 Branched chain amino acids regulate mammary function and embryo development. BCAA play a vital role in mammary function and
embryo development mainly in the synthesis of other conditional amino acids and activation of the mTOR signaling pathway
Zhang et al. Journal of Animal Science and Biotechnology (2017) 8:10 Page 6 of 12
kg body weight to breast-fed neonates improves intes-
tinal development and increases the expression of neu-
tral amino acid transporters (ATB
0,+
,B
0
AT1 and b
0,+
AT)
[90]. In a study conducted in our lab, we demonstrated
that meeting BCAA requirement (supplementing
0.1% L-Leu, 0.34% L-Val, and 0.19% L-Ile in 17% crude
protein diet) is necessary for maintaining intestinal
health and amino acid transporter expression [63], the
latter of which might be through the PI3K/Akt/mTOR
and ERK signaling pathways [91]. Interestingly, we found
that leucine not only increased the expression of neutral
amino acid transporters (ASCT2, rBAT and 4F2hc), but
also cationic amino acid transporters (CAT1) which em-
phasizes the importance of BCAA in intestinal nutrient
absorption.
Besides regulating intestinal amino acid transporter
expression, BCAA also have an intimate connection with
other intestinal functions. Elevating dietary leucine level
from 1.37% to 2.17% enhanced the intestinal develop-
ment of broilers through the mTOR signaling pathway
[92]. Concurrently, Mao et al.[93] demonstrated that
extra dietary 1% leucine supplementation alleviated a de-
crease in mucin production and goblet cell numbers in
the jejunal mucosa of weaned pigs, which possibly oc-
curs via activation of mTOR signaling. Similarly, the role
of leucine (increasing leucine level from 0.71% to 1.33%)
in maintaining gut health (enhancing tight junction) was
also demonstrated in fish [94].
BCAA can be utilized by bacteria in the lumen of the
gut [95]. Based on the 24-h disappearance rates of amino
acids in different intestinal segments, Dai et al.[95] di-
vided AA into three groups (high, medium or low dis-
appearance rate groups), and found that leucine belongs
to the high disappearance rate group while isoleucine
and valine belong to the medium disappearance rate
group. This is important evidence that BCAA participate
in bacterial metabolism indicating they might participate
in the regulation of intestinal microbial species and
diversity. More research needs to be done to elucidate
the detailed changes in these microbial species.
Collectively, most of the studies still focus on the func-
tions of leucine but not valine or isoleucine in the intes-
tine. However, the high expression of BCAT and BCKT
in the intestine indicates the strong connection between
BCAA and intestinal function.
BCAA and immune function
People noticed the effects of BCAA on the immune sys-
tem 10 yr ago because immune cells oxidize BCAA as
fuel sources and incorporate BCAA as the precursors
for the synthesis of new immune cells, effector mole-
cules, and protective molecules [9]. Lack of BCAA in
the diet impairs many aspects of immune function and
increases susceptibility to pathogens. A recent study
showed that a daily 12 g BCAA oral supplementation
improved phagocytic function of neutrophils and NK
activity of lymphocytes in cirrhotic patients [96]. After
a BCAA enriched solution was infused into patients
with rectal cancer, their immune status was improved
with increased CD4+, CD4+/CD8+ and IL-2R [97].
Similarly, 12 g/d of BCAA (6 g/d L-Leu, 2 g/d L-Iso
and 4 g/d L-Val) supplementation blunted the neutrophil
response to intense cycling training, which might benefit
immune function during a prolonged cycling season [98].
In animal husbandry, supplementing BCAA (0.1% L-Leu,
0.34% L-Val, and 0.19% L-Ile) in a 17% crude protein diet
was shown to improve intestinal immune defense func-
tions with an increase of intestinal immunoglobulins
(IgA and sIgA) in weaned piglets [99]. In contrast, some
studies also reported that BCAA mixture supplementation
(600 mg/kg body weight/day, consist of 46% leucine, 28%
valine, and 23% isoleucine) could not ameliorate the
impaired function of macrophages induced by strenuous
exercise in rats [100].
In recent years, there has been growing interest in the
role of isoleucine, leucine and valine in immune function
(Table 1). Notably, concentrations of isoleucine have a
strong correlation with the excretion of β-defensin. 25
or 50 μg/mL isoleucine increases the mRNA and protein
expressions of β-defensin 1, 2 and 3 in IPEC-J2 cells
[101]. Also, treating patients with 250 μg of intratracheal
L-isoleucine every 48 h is considered as a novel im-
munotherapy in tuberculosis as it induced a significant
increase of β-defensins 3 and 4 associated with de-
creased bacillary loads and tissue damage [102]. This in-
ducing function of isoleucine might be associated with
the G-Protein Coupled Receptor and ERK signaling
pathways [103]. Additionally, proximally 2% dietary iso-
leucine could enhance intestinal immunity in juvenile
Table 1 Branched chain amino acids and immune function
Amino acid Regulation of immune function
BCAA Mix ●
↑fuel sources for immune cells
●
↑immune function of neutrophils and lymphocytes
●
↑CD4+, CD4+/CD8+
●
↑intestinal immunoglobulins
Isoleucine ●
↑excretion of β-defensin
Leucine ●
↑regulation of innate and adaptive immune responses
●
↑pro-inflammatory cytokines ↓anti-inflammatory cytokines
Valine ●
↑dendritic cell function
●
↑pro-inflammatory cytokines ↓anti-inflammatory cytokines
Zhang et al. Journal of Animal Science and Biotechnology (2017) 8:10 Page 7 of 12
Jian carp and innate immunity in olive flounder [104,
105]. Similar functions in regulation of the immune re-
sponse and antioxidant status in the head kidney were
also observed in fish fed about 1.3% isoleucine [106]. In
contrast to isoleucine, leucine regulates the immune sys-
tem mainly through the mTOR signaling pathway.
mTOR plays a vital role in the regulation of the innate
and adaptive immune responses and also several
immune functions like promoting differentiation, activa-
tion, and function in T cells, B cells and antigen-
presenting cells [107, 108]. For instance, a reasonable
dose of leucine (40 mg/mL) provides enhanced protect-
ive immunity against mucosal infection with herpes sim-
plex virus type 1 [109]. Leucine deficiency could impair
the immune status, up-regulate pro-inflammatory cyto-
kines and down-regulate anti-inflammatory cytokines of
grass carp (Ctenopharyngodon idella) by the NF-κB and
TOR signaling pathways which was reversed by
optimum leucine supplementation [94]. Recently, an
in vitro experiment found that an increased extracellular
concentration of BCAA, especially valine (800 nmol/mL),
could improve the dendritic cell function in cirrhotic pa-
tients [110]. In addition, valine deficiency (less than
1.45%) decreased growth and intestinal immune status
in young grass carp (Ctenopharyngodon idella) by in-
creasing pro-inflammatory cytokines (IL-8 and TNF-α)
and decreasing anti-inflammatory cytokines (IL-10 and
TGF-β1) which might be caused by changes in the NF-κB
and mTOR signaling pathways [111]. However, some
studies found there was little effect of valine (increase
from 0.64% to 0.87%) on innate or adaptive immunity
for broilers [112]. Although still unclear, all these studies
areevidencethatBCAAmayfunctioninimproving
health and preventing infectious diseases in animals and
humans by regulating the immune system.
BCAA, a biomarker for early pathogenesis of chronic
diseases
Obesity is strongly associated with the risk of developing
a number of chronic diseases including diabetes, gall-
stones, hypertension, heart disease and stroke [113]. Sci-
entists have attempted to find biomarkers which can
connect the incidence of these diseases via metabolo-
mics. Alterations in their metabolism may play a vital
role in the early pathogenesis for humans. Recently, the
relationship between metabolomics and obesity (insulin-
resistant) was revealed by a series of studies [114].
Newgard et al.[15] found that some major components
obtained from obese (insulin-resistant) versus lean
(insulin sensitive) subjects were different, including
long-chain fatty acids, ketone metabolites and medium-
chain acylcarnitines, but surprisingly, the component
which was most explicitly associated with insulin sensi-
tivity was not the lipid related components mentioned
above, but rather was comprised of the BCAA, the aro-
matic amino acids, C3 and C5 acylcarnitines (Fig. 1), as
well as glutamate and alanine. Concurrently, recent
metabolomics conducted with 2,422 normoglycemic in-
dividuals for 12 yr showed a strong association between
metabolite profiles (branched-chain and aromatic amino
acids) and future diabetes [115]. In addition, subsequent
reports showed that leucine and isoleucine levels (but
not valine) are correlated with insulin resistance and
blood glucose levels which indicates that analyzing
BCAA separately might be better for understanding the
association between BCAA and obesity [116]. In study
involving twins, BCAA catabolism of the obese was
down-regulated compared with lean co-twins in the
adipose tissue [117], which could be caused by a decrease
of BCATm and BCKD E1αprotein concentrations (first
two enzymatic steps of BCAA catabolism)[118]. Studies in
animals also strongly support the findings in humans.
BCAA metabolism is weak in diabetic mice compared
with non-diabetic mice [119]. In addition, high running
capacity rats express more BCAA degradation and fatty
acid metabolism genes than low running capacity rats
[120]. Taken together, elevated BCAA levels in blood
(decreased BCAA catabolism) are associated with obesity,
diabetes, and other risk factors for metabolic diseases and
are a good biomarker for early pathogenesis of these
diseases in humans.
Conclusion and perspectives
BCAA are essential amino acids for animals and humans
not only because they cannot be synthesized in the body
but also because they display remarkable metabolic and
regulatory roles. In humans and animals, BCAA (espe-
cially leucine) enhance protein synthesis through the
mTOR signaling pathway and now are considered as
feed additives to regulate meat quality and are used as
performance-enhancing supplements for body builders
and fitness enthusiasts. Recently, novel metabolic and
physiological functions of BCAA have been reported by
scientists. BCAA are metabolic regulators not only in
protein synthesis but also in lipid and glucose metabol-
ism. They enhance mammary health, increase milk qual-
ity and help in early embryo implantation and
development. They improve gut health and its local
amino acid transporting ability. They enhance immunity
by increasing the expression of β-defensin, up-regulating
pro-inflammatory cytokines and down-regulating anti-
inflammatory cytokines. Finally, they are biomarkers for
early detection of chronic diseases like diabetes and
insulin resistance in humans.
A growing body of evidence suggests that food has
specific direct and indirect actions to activate intestinal
receptors like a cocktail of ‘hormones’[121]. This
Zhang et al. Journal of Animal Science and Biotechnology (2017) 8:10 Page 8 of 12
activation can increase the secretion of GI tract hor-
mones like peptide YY (PYY), glucagon-like peptide 1
(GLP-1) and cholecystokinin (CCK) [122]. There are var-
iety of receptors in the GI tract for amino acids which
have been discovered such as T1R1/T1R3, CaSR,
GPCR6A and mGluR [123]. The activation of these re-
ceptors might participate in the regulation of food in-
take, proliferation of GI cells, small intestinal motility
and neural reflexes [124]. However, the specific recep-
tors for BCAA are still a mystery and wait to be discov-
ered. Knowing the receptors of BCAA is vital for a
better understanding of the physiological roles of BCAA.
In the future, with the help of high throughput func-
tional genomics, metabolomics, and proteomics, the
underlying functions of BCAA in gene, protein expres-
sion and metabolic regulation will be revealed. The ef-
fects of the BCAA on microbe numbers can be analyzed
by 16S targeted sequencing and metagenomic sequen-
cing. All of these techniques will help in interpreting the
complex and inconsistent results obtained to date and
largely expand our vision of the novel functions of
BCAA in humans and animals.
Acknowledgements
The author would like to thank Dr. M. A. Brown and Dr. P. A. Thacker for their help.
Funding
This work was supported by the National Key Basic Research Program (S.Y.Q.,
Grant Number 2012CB124704).
Availability of data and materials
Not applicable.
Authors’contributions
SZ initiated the idea, the scope, and the outline of this review paper. SZ, MR,
XM, SQ and XZ studied and analyzed all of the publications cited in this
paper and were involved in the manuscript preparation. XZ conducted the
final editing and proofreading. All authors read and approved the final
manuscript.
Competing interests
The authors declare that they have no competing interests.
Consent for publication
Not applicable.
Ethics approval
All procedures used in experiments of our lab were performed in
accordance with the China Agricultural University Animal Care and Use
Committee guidelines (ID: SKLAB-B-2010-003).
Author details
1
State Key Laboratory of Animal Nutrition, College of Animal Science and
Technology, China Agricultural University, No.2 Yuanmingyuan West Road,
Haidian District, Beijing 100193, People’s Republic of China.
2
College of
Animal Science, South China Agricultural University, Wushan Avenue, Tianhe
District, Guangzhou 510642, People’s Republic of China.
3
College of Animal
Science, Anhui Science & Technology University, No. 9 Donghua Road,
Fengyang 233100, Anhui Province, People’s Republic of China.
4
Animal
Nutrition Institute, Key Laboratory of Animal
Disease-ResistanceNutrition,Ministry of Education, Sichuan
AgriculturalUniversity, Ya’an, Sichuan, China.
Received: 26 July 2016 Accepted: 27 December 2016
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