Download Branched-chain amino acids improve glucose

Am J Physiol Gastrointest Liver Physiol 288: G1292–G1300, 2005.
First published December 9, 2004; doi:10.1152/ajpgi.00510.2003.
Branched-chain amino acids improve glucose metabolism
in rats with liver cirrhosis
Shinobu Nishitani, Kenji Takehana, Shoji Fujitani, and Ichiro Sonaka
Pharmaceutical Research Laboratories, Ajinomoto Co., Inc., Kawasaki-ku, Kawasaki-shi, Japan
Submitted 4 December 2003; accepted in final form 29 November 2004
mammalian target of rapamycin; glycogen synthase; GLUT4;
GLUT1; rat model of carbon tetrachloride-induced cirrhosis
that cirrhosis is associated with
impaired glucose metabolism, with the majority of patients
(60 – 80%) being glucose intolerant and 10 –15% eventually
developing overt diabetes mellitus (24). The characteristics of
glucose intolerance in cirrhotic patients are unusual, because
although these patients exhibit fasting hypoglycemia, they also
have postprandial hyperglycemia and continuous hyperinsulinemia (11). Despite being hyperinsulinemic, many cirrhotic individuals have impaired glucose tolerance or are overtly diabetic.
Hyperinsulinemia in the face of hyperglycemia suggests the
presence of insulin resistance (6, 26, 30). In this condition, it is
believed that postprandial glucose uptake is not carried out
promptly in insulin-sensitive tissues, such as skeletal muscle
and adipose tissue, and that glycogen synthesis is not promoted
IT HAS LONG BEEN RECOGNIZED
Address for reprint requests and other correspondence: S. Nishitani and K.
Takehana, 1-1, Suzuki-cho, Kawasaki-ku, Kawasaki-shi 210-8681, Japan (Email: [email protected], [email protected]).
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in skeletal muscle or liver. Indeed, there is considerable evidence that liver and skeletal muscle glycogen content is lower
in cirrhotic patients than in healthy individuals (29). Under
these conditions, even the control of blood glucose during
hunger is difficult, with the characteristics of glucose intolerance described above being found in cirrhotic patients. In
skeletal muscle and liver, glycogen synthesis is very important
and is related directly to blood glucose control. More importantly, it has been reported that glucose intolerance in cirrhotic
patients enhances the risk of hepatic insufficiency (4). Two
studies in Italy and Japan showed that long-term oral supplementation with branched-chain amino acid (BCAA) in advanced cirrhosis was useful for prevention of progressive
hepatic failure, inasmuch as it improved surrogate markers and
perceived health status (8a, 9a, 12, 18). In Japan, pharmacological supplementation of BCAA is used widely to ameliorate
hypoalbuminemia in patients with decompensated liver cirrhosis. This supplementation involves the use of LIVACT granules administered in four doses, three times a day after meals
to provide a 12 g/day dose of BCAA. The weight ratio of
leucine to isoleucine to valine in the granules is 2:1:1.2. In the
present study, we chose to administer a dose of 1.5 g/kg BCAA
to rats, inasmuch as our previous experiments showed that this
dose of BCAA in rats achieved and maintained almost the
same plasma concentration of BCAA as that measured in
patients given 4 g of BCAA mixture.
In a recent report, we showed that leucine stimulated glucose
uptake in isolated soleus muscle from normal rats under insulin-free conditions (23), whereas in a study in a myoblast cell
line carried out by other investigators, glycogen synthase (GS)
was activated by the mammalian target of rapamycin (mTOR)
(25). In addition, there is evidence in cultured human muscle
cells that total amino acid level regulates GS activity via
mTOR (2). Thus we assumed that BCAA may regulate glucose
metabolism in cirrhosis, a condition in which blood BCAA
levels are decreased. In this study, we examined whether
BCAA regulated glucose metabolism in a rat model of cirrhosis, an animal model that has been shown to be glucose
intolerant (20, 21). To achieve this objective, we investigated
the subcellular location of glucose transporters and the activity
of glycogen synthesis in skeletal muscles, in which it is
possible to carry out some of the substituted functions of
glucose metabolism undertaken by the liver.
MATERIALS AND METHODS
Materials. The mTOR inhibitor rapamycin was purchased from
Wako Pure Chemicals (Osaka, Japan). All other chemicals were of the
best grade commercially available.
The costs of publication of this article were defrayed in part by the payment
of page charges. The article must therefore be hereby marked “advertisement”
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
0193-1857/05 $8.00 Copyright © 2005 the American Physiological Society
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Nishitani, Shinobu, Kenji Takehana, Shoji Fujitani, and
Ichiro Sonaka. Branched-chain amino acids improve glucose metabolism in rats with liver cirrhosis. Am J Physiol Gastrointest Liver
Physiol 288: G1292–G1300, 2005. First published December 9, 2004;
doi:10.1152/ajpgi.00510.2003.—It is well established that impaired
glucose metabolism is a frequent complication in patients with hepatic
cirrhosis. We previously showed that leucine, one of the branchedchain amino acids (BCAA), promotes glucose uptake under insulinfree conditions in isolated skeletal muscle from normal rats. The aim
of the present study was to evaluate the effects of BCAA on glucose
metabolism in a rat model of CCl4-induced cirrhosis (CCl4 rats). Oral
glucose tolerance tests were performed on BCAA-treated CCl4 rats. In
the CCl4 rats, treatment with leucine or isoleucine, but not valine,
improved glucose tolerance significantly, with the effect of isoleucine
being greater than the effect of leucine. Glucose uptake experiments
using isolated soleus muscle from the CCl4 rats revealed that leucine
and isoleucine, but not valine, promoted glucose uptake under insulinfree conditions. To clarify the mechanism of the blood glucoselowering effects of BCAA, we collected soleus muscles from BCAAtreated CCl4 rats with or without a glucose load. These samples were
used to determine the subcellular location of glucose transporter
proteins and glycogen synthase (GS) activity. Oral administration of
leucine or isoleucine without a glucose load induced GLUT4 and
GLUT1 translocation to the plasma membrane. GS activity was
augmented only in leucine-treated rats and was completely inhibited
by rapamycin, an inhibitor of mammalian target of rapamycin. In
summary, we found that leucine and isoleucine improved glucose
metabolism in CCl4 rats by promoting glucose uptake in skeletal
muscle. This effect occurred as a result of upregulation of GLUT4 and
GLUT1 and also by mammalian target of rapamycin-dependent activation of GS in skeletal muscle. From these results, we consider that
BCAA treatment may have beneficial effects on glucose metabolism
in cirrhotic patients.
BRANCHED-CHAIN AMINO ACIDS IMPROVE GLUCOSE METABOLISM
RESULTS
Effects of BCAA on blood glucose and plasma insulin levels
in CCl4 rats. After 17 h of fasting, the CCl4 cirrhotic rats were
subjected to an OGTT, with measurement of blood glucose and
insulin concentrations. We chose a high-dose glucose load of 4
g/kg, inasmuch as we found previously that this was the
optimal dose for investigation of muscle glycogen synthesis
after 3 days of fasting (data not shown). We checked the
optimal timing for BCAA administration in OGTT experiments and concluded that 60 min after the glucose load
produced the best results. Blood glucose level in the control
group peaked at 0 min, that is, 1 h after the glucose load. The
level remained elevated for a further 120 min, despite continuing release of insulin (Fig. 1, A and D). These results indicated
a pathological phenotype of impaired glucose tolerance in the
CCl4 cirrhotic rats. In the Leu group, blood glucose levels
peaked at the same time as in the Cont group, but 30 min after
leucine administration, the level decreased significantly (Fig.
1A). Administration of leucine did not appear to stimulate
additional insulin release, inasmuch as insulin levels were
similar in the Cont and Leu groups at all time points, except
120 min (Fig. 1B).
In the next series of experiments, we examined whether
isoleucine or valine affected glucose metabolism in cirrhotic
rats. Blood glucose levels in the Ile group were decreased
significantly 30 min after administration compared with the
Cont group, whereas administration of valine had almost no
effect (Fig. 1D). Plasma insulin levels were similar in the Ile,
Val, and Cont groups, whereas isoleucine or valine administration did not stimulate insulin release further (Fig. 1E).
Because leucine and isoleucine improved glucose levels without additional insulin secretion (Fig. 1, B and E), we assumed
that these amino acids improved glucose metabolism without
additional insulin secretion.
Comparison of effects of leucine and isoleucine on glucose
tolerance. The findings described above showed that leucine
and isoleucine improved glucose tolerance in CCl4 rats,
whereas valine did not. To compare the blood glucose-lowering effects of leucine and isoleucine, we performed another
series of OGTTs on the CCl4 rats. In these experiments, we
chose a 2 g/kg glucose load to clarify the difference between
the effect of leucine and the effect of isoleucine. At 30 min
after administration of leucine or isoleucine, blood glucose
levels in both groups were significantly decreased. Although
blood glucose levels in the Leu group remained unchanged
from 30 to 120 min, levels in the Ile group decreased continuously even after 60 min (Fig. 2A). Changes in plasma insulin
levels were similar in the three groups (Fig. 2B). These results
suggested that glucose levels were decreased to a greater extent
by isoleucine than by leucine (Fig. 2C).
Glucose uptake in soleus muscle of CCl4 rats treated with
BCAA. The blood glucose-lowering activities of leucine and
isoleucine are not mediated by additional insulin secretion.
Therefore, we assumed that the main action of BCAAs in
tissues is to stimulate glucose consumption. Muscle is one of
the major glucose-consuming tissues in the body. We also
showed previously that, in normal rats, leucine directly promotes glucose uptake in isolated muscle (23). The following
series of experiments were performed to examine the effects of
leucine and isoleucine on glucose uptake in isolated soleus
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Animals. The following experimental protocol was reviewed and
approved by the Animal Care Committee of Ajinomoto Co., Inc. Male
Sprague-Dawley rats, 5 wk of age, were obtained from Charles River
Japan (Yokohama, Japan). They were maintained in individual cages
in an air-conditioned room (24 ⫾ 1°C ) with a 12:12-h light-dark
cycle (lights on from 0700 to 1900). The animals were fed a stock
pellet diet (CRF-1, Oriental Yeast, Tokyo, Japan). The CCl4 rats were
prepared according to the standard method of Kajiwara et al. (12).
After 15 wk, plasma albumin, total bilirubin, and alanine aminotransaminase (ALT) levels were determined. A kit was used to measure
albumin (Wako Pure Chemicals), and a Dri Chem 5500 was used to
measure total bilirubin and ALT (Fuji Medical System, Tokyo,
Japan). Rats with an albumin value of 2.8 –3.3 g/dl, total bilirubin
⬎1.5 mg/dl, and ALT activity ⬎100 U/l were selected for the
experiments.
Oral glucose tolerance test and insulin measurement with BCAA
administration. After 17 h of fasting, the CCl4 rats (450 –550 g body
wt) were given a compulsory oral 2 or 4 g/kg glucose load at ⫺60 min
followed at ⫺30 or 0 min by gavage administration of saline (Cont
group) or 1.5 g/kg leucine (Leu group), isoleucine (Ile group), or
valine (Val group). Blood glucose concentration was measured at
⫺60, 0, 30, 60, 90, 120, and 180 min using a Dri Chem 5500. Insulin
concentration was measured at the same time points using an insulin
kit (Morinaga, Tokyo, Japan), except for the 180-min sample.
Glucose uptake in isolated soleus muscle. CCl4 rats were killed by
decapitation, and the soleus muscles were isolated immediately from
both legs. Glucose uptake in incubated soleus muscles was determined
by a procedure we described previously (23).
Preparation of plasma and intracellular membranes from skeletal
muscle tissue. Plasma membranes from stock samples of soleus and
gastrocnemius muscles (6 g weight) of several CCl4 rats, obtained 30
min after compulsory oral administration of 1.5 g/kg of each BCAA
(leucine, isoleucine, valine) or BCAA as LIVACT compositions
(2:1:1.2 leucine-isoleucine-valine) or 2 g/kg glucose or saline, were
prepared according to previously described methods (15, 22).
GLUT4 and GLUT1 immunoblotting. Rabbit anti-GLUT4 or antiGLUT1 (Chemicon International) was used to detect GLUT4 and
GLUT1 in the plasma membrane. The membrane fractions were also
immunoblotted by Na⫹-K⫹-ATPase antibody (American Research
Products). The bands were quantified using Scion Image software and
standardized against the intensity of the Na⫹-K⫹-ATPase bands.
Glycogen content and GS assay. The CCl4 rats were deprived of
food for 3 days and then selected randomly for injection via the tail
vein with 0.75 mg/kg rapamycin (Wako Pure Chemicals) or an equal
volume of vehicle [saline and 2% (vol/vol) ethanol] as described
previously (1). A compulsory oral glucose load of 4 g/kg was
administered 60 min before 1.5 g/kg of leucine, isoleucine, or saline.
After a further 60 min, the soleus muscles were isolated under ether
anesthesia for use in subsequent GS and p70 S6 kinase assays. The
amount of BCAA administered was equivalent to the average amount
of amino acids consumed by rats of this strain during 24 h of free
access to food (9) and resulted in plasma total BCAA concentrations
of ⬃2 mM. The CCl4 rats were deprived of food for 3 days but
allowed free access to water to deplete skeletal muscle glycogen
content, as described previously (19). Soleus muscles of the rats were
hydrolyzed with KOH, and the glycogen content of this preparation
was determined as described previously (19). A modified filter paper
method was used to measure GS activity as described previously, with
the results expressed as mean activity per total activity (8).
p70 S6 kinase assay. The activity of p70 S6 kinase was measured
in isolated soleus muscle samples according to the method of Hara et
al. (10).
Statistics. Values are means ⫾ SE. Differences in mean values
were analyzed by Student’s t-test and one-way ANOVA followed by
Dunnett and Tukey-Kramer analyses. P ⬍ 0.05 was considered
significant.
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Fig. 1. Effects of branched-chain amino acid (BCAA)
administration on oral glucose tolerance test (OGTT) in
rats in which cirrhosis was induced by CCl4. A and D:
after a 17-h fast, CCl4 rats were treated with 4 g/kg
glucose at ⫺60 min and 1.5 g/kg BCAA (Leu, Ile, and
Val) at 0 min. Control (Cont) group was treated with
saline (vehicle for amino acids) at ⫺60 min. Blood
glucose concentration was measured at ⫺60, 0, 30, 60,
90, and 120 min. B and E: insulin concentration was
measured at the same time points, and mean area under
the curve (AUC) of plasma insulin was calculated. C
and F: mean ratio of glucose AUC to insulin AUC
demonstrates effect on insulin sensitivity. Values are
means ⫾ SE for 5 rats. *P ⬍ 0.05 vs. Cont (Student’s
t-test).
muscle from CCl4 rats and also to elucidate the mechanism of
this glucose-lowering effect. Figure 3 demonstrates that
leucine and isoleucine, but not valine, promoted glucose uptake
in isolated soleus muscle from CCl4 rats. Although the blood
glucose-lowering effects of leucine and isoleucine seemed to
improve insulin sensitivity (Figs. 1, C and F, and 2C), which
was indicated as glucose-to-insulin area under the curve ratio
in OGTT experiments, both amino acids were able to stimulate
glucose uptake in the absence of insulin. These results suggested that isoleucine and leucine would act directly to stimulate glucose uptake as well as insulin secretion.
Effects of BCAA administration on translocation of GLUT1
and GLUT4 to the plasma membrane. On the basis of the
above finding, we next examined whether orally administered
BCAAs had any direct effects on the muscle of cirrhotic rats.
Transporter-mediated uptake of glucose is the rate-limiting
determinant of glucose utilization in skeletal muscle (3).
GLUT4 is the most abundant glucose transporter isoform in
skeletal muscle and adipose tissue (16, 27, 28) and is responsible for the majority of insulin-dependent glucose uptake in
these tissues (14). It has long been recognized that insulin
promotes the rapid translocation of GLUT4 from intracellular
membrane compartments to the plasma membrane (16, 27, 28).
GLUT1 protein is expressed in almost all tissues but is a minor
isoform in skeletal muscle. To examine whether leucine and
isoleucine have any effects on translocation of GLUT4 and
GLUT1, we used immunoblotting to measure the amount of
these transporters in skeletal muscle membrane fractions. The
data were standardized against activity for Na⫹-K⫹-ATPase, a
protein localized constitutively at the plasma membrane (see
Fig. 5C). Muscle samples were isolated from rats treated with
BCAA, glucose, and BCAA ⫹ glucose. Additional adminis-
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trations of BCAAs augmented the glucose-induced plasma
membrane translocation of GLUT4 in skeletal muscle of CCl4
rats (Fig. 4). Moreover, oral administration of leucine or
isoleucine alone, but not valine, promoted translocation of the
GLUT4 protein to the plasma membrane, similar to that observed with administration of glucose (Fig. 5A). GLUT1 translocation was also promoted by administration of leucine, isoleucine, or glucose alone (Fig. 5B). The plasma insulin levels
increased significantly after administration of glucose or
leucine, but not isoleucine (Fig. 5D).
Glycogen content in soleus muscle in normal and CCl4 rats.
Skeletal muscle is one of the main tissues for storing energy in
the body, with glycogen being synthesized from excess glucose
as a future fuel reserve. It is well known that cirrhotic patients
have low glycogen stores, not only in liver but also in muscle
(11, 24). In animals receiving normal feed, we found that
glycogen content in isolated soleus muscles and liver was
significantly lower in CCl4 than in normal rats (Fig. 6A). The
next experiments were performed with the aim of evaluating
the effects of BCAA on glycogen synthesis. As shown above,
leucine and isoleucine acted directly on muscle to promote
glucose uptake and improve glucose intolerance after an oral
glucose load in cirrhotic rats. Glucose uptake and glycogen
synthesis, a major use of glucose, are thought to be essential for
normal postprandial glucose homeostasis. We measured GS
activity in isolated muscles from CCl4 rats fasted for 3 days. At
⫺60 min these animals were given a glucose load, at time 0
leucine or isoleucine was administered, and soleus muscles
were isolated at 60 min, when the difference in blood glucose
levels between controls and the BCAA group was greatest. GS
activity in isolated soleus muscle from the CCl4 rats was
upregulated significantly by the administration of leucine, but
not isoleucine (Fig. 6B). We also found that glycogen content
in soleus muscle tended to be greater in the Leu than in the
Cont group (data not shown).
Effects of rapamycin on GS and p70 S6 kinase activity in
soleus muscle and blood glucose and plasma insulin levels
after leucine administration. To elucidate the mechanism of
leucine-induced GS activity (Fig. 6B), we investigated mTOR
signaling, a pathway that is thought to be a cellular sensor for
amino acids. Although leucine stimulated GS activity, it also
induced strong phosphorylation of p70 S6 kinase, a downstream target of mTOR protein kinase (Fig. 7, A and B). GS
activity and p70 S6 kinase phosphorylation were inhibited by
pretreatment with rapamycin. Blood glucose and plasma insu-
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Fig. 2. Comparison of effects of leucine and isoleucine
on OGTT in CCl4 rats. After a 17-h fast, CCl4 rats were
treated with 2 g/kg glucose at ⫺60 min and 1.5 g/kg Leu
or Ile at ⫺30 min. Cont group was treated with saline as
the vehicle at ⫺30 min. Blood glucose (A) and insulin
(B) concentrations of normal rats were measured at
⫺60, 0, 30, 60, 90, and 120 min, and mean AUC of the
plasma insulin was calculated. C: mean ratio of glucose
AUC to insulin AUC demonstrates effect on insulin
sensitivity. Values are means ⫾ SE of 5 rats. *P ⬍ 0.05
compared with Cont (Student’s t-test).
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lin concentrations were not altered by pretreatment with rapamycin (Fig. 7, C and D).
DISCUSSION
BCAA mixtures have been used extensively in Japan as a
prescription drug for the treatment of hypoalbuminemia in
patients with decompensated liver cirrhosis. Impaired glucose
metabolism occurs frequently in patients with cirrhosis and is
thought to be among the risk factors that contribute to the
serious complications associated with the disorder. The present
studies were planned and carried out to evaluate whether
BCAA supplementation improved the impaired glucose metabolism associated with cirrhosis. These investigations were
based on findings from several clinical studies that BCAA
supplementation improved insulin tolerance and glucose tolerance in patients with liver cirrhosis (10a, 11a, 26b).
We investigated the effects of BCAA on glucose metabolism
in rats with CCl4-induced cirrhosis and found that leucine and
isoleucine had characteristic pharmacological effects on skeletal muscle that led to improved glucose metabolism. Our data
showed that, after a glucose load in CCl4 rats, administration of
leucine or isoleucine reduced blood glucose level without
increasing insulin secretion. A possible mechanism to explain
these changes is enhancement of glucose uptake via upregulation of GLUT4 and GLUT1 translocation (Figs. 4 and 5). We
emphasize that this is the first report that isoleucine promotes
in vivo glucose uptake in isolated soleus muscle under pathological conditions (Fig. 3), presumably by its ability to promote
translocation of glucose transporter proteins to the plasma
membrane in the absence of additional insulin release (Fig. 5).
However, another recent report showed that isoleucine promoted glucose uptake in vitro in the myoblast cell line C2C12
(7a). We assume for the reasons listed below that this increase
in glucose uptake is mediated by a novel signal independent of
Fig. 4. Additive effects on GLUT4 translocation to plasma membrane by
orally administered BCAA and glucose in skeletal muscle of CCl4 rats. After
17 h of fasting (Fast), BCAA (1.5 g/kg) and vehicle (saline) were administered
with a 2 g/kg glucose (Glc) load. Muscle samples were collected at 60 min on
the OGTT graph. Protein (4.2 ␮g) of each membrane fraction was loaded, and
GLUT4 expression was detected by immunoblotting.
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Fig. 3. Effects of BCAA on glucose uptake in isolated soleus muscle from
CCl4 rats. Soleus muscle isolated from CCl4 rats after 17 h of fasting (Fast)
was incubated in tubes with 2 mM Leu, Ile, or Val or saline (None) for 20 min
and then incubated for another 20 min with double-labeled glucose as described previously (23). *P ⬍ 0.05 vs. None.
mTOR signaling. First, we showed previously that leucine
upregulates glucose uptake directly in isolated skeletal muscle
in a rapamycin-resistant manner (23), with this action being
blocked by phosphatidylinositol 3-kinase (PI3K) and PKC
inhibitors. In our previous experiment, the effect of leucine was
blocked completely by LY-294002 (a specific inhibitor of
PI3K) and GF-109203X (a specific inhibitor of PKC). These
results suggest that leucine promotes glucose uptake in skeletal
muscle via the PI3K and PKC pathways. The present study
demonstrates that isoleucine and leucine have similar effects
on glucose uptake and translocation of GLUT4 and GLUT1.
We anticipate that GLUT4 translocation induced by BCAAs
may be mediated by PI3K and atypical PKC signals, because
both of these factors are thought to be involved in the translocation of GLUT4 (5, 7, 23). Using isolated soleus muscles
from CCl4 rats, we observed results similar to those seen in
normal rats (data not shown). Additionally, we confirmed
previously that isoleucine and leucine promoted GLUT4 translocation without activation of Akt phosphorylation, a wellknown reaction downstream of PI3K signaling that leads to
induction of GLUT4 translocation by insulin. Therefore, further detailed analyses are required to clarify how BCAAs
induce the signaling for GLUT4 translocation (unpublished
observations). These additional studies also need to examine
other energy status-regulated signals, such as AMP kinase. On
the other hand, there are several reports indicating that insulin
and a change in energy status result in upregulation of GLUT1
mRNA and translocation of the transporter to the plasma
membrane (13, 23a, 26a, 31). Inasmuch as BCAAs have
similar effects, it is possible that these compounds act as novel
signal inducers of an alternative pathway. The mechanism of
GLUT1 induction by leucine and isoleucine, however, remains
unclear and requires clarification in future studies.
Our data also suggest that there may be another mechanism
to account for the effects of leucine on glucose metabolism in
skeletal muscle, namely, promotion of glycogen synthesis via
activation of mTOR signaling (Figs. 6 and 7). We found that
GS activity in soleus muscles was augmented by leucine, but
not by isoleucine, and was inhibited completely by pretreatment with rapamycin (Fig. 7, A and B). We assume that
differences in the potential of leucine and isoleucine to activate
BRANCHED-CHAIN AMINO ACIDS IMPROVE GLUCOSE METABOLISM
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Fig. 6. Effects of orally administered Leu and Ile on glycogen
synthase (GS) activity in CCl4 rats after glucose load. A:
glycogen content in isolated soleus muscle and liver measured
in normal and CCl4 rats. Values are means ⫾ SE of 5 rats. *P ⬍
0.05 compared with normal rats (Student’s t-test). B: after 3
days of fasting, CCl4 rats were treated with 4 g/kg glucose at
⫺60 min and 1.5 g/kg Leu, Ile, or saline (Cont) at 0 min. After
60 min, soleus muscles were isolated from each of the CCl4
rats, and GS activity was measured. *P ⬍ 0.05 compared with
Cont (Student’s t-test).
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Fig. 5. Effects of single BCAAs on GLUT1 and GLUT4
translocation to plasma membrane in skeletal muscle of CCl4
rats. After 17 h of fasting, 1.5 g/kg BCAA and 2 g/kg glucose
or vehicle (saline) were administered at 0 min. After 60 min,
skeletal muscles were isolated from the rats. A and B: immunoblots for GLUT4 and GLUT1, (4.2 and 7.5 ␮g of protein,
respectively). Each band was detected in 3 rat samples and
scanned and quantified by Scion Image beta 4.02. C: Na⫹-K⫹ATPase was detected as a plasma membrane marker (protein
content of each application ⫽ 7.5 ␮g). D: plasma insulin
concentration at the time of soleus muscle collection. *P ⬍ 0.05
compared with Fast (Dunnett’s multiple comparison test).
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Fig. 7. Effect of rapamycin on blood glucose level, plasma insulin, and soleus
muscle GS activity after Leu administration to CCl4 rats subjected to an
OGTT. CCl4 rats fasted for 3 days were selected randomly for injection via tail
vein with 0.75 mg/kg rapamycin (Rap) or an equal volume of vehicle (saline,
2% vol/vol ethanol; Fast). In Leu and Cont groups, oral administration of 4
g/kg glucose at ⫺60 min was followed by administration at 0 min of 1.5 g/kg
Leu or saline. After 60 min, blood was collected for measurement of blood
glucose and plasma insulin, and soleus muscle was isolated under ether
anesthesia. Samples were used to measure GS and p70 S6 kinase phosphorylation. A: p70 S6 kinase phosphorylation in Cont, Leu, and Rap groups. B: GS
activity/total activity in Fast, Cont, Leu, and Rap groups. C: blood glucose in
Fast, Cont, Leu, and Rap groups. D: insulin concentration in Fast, Cont, Leu,
and Rap groups. Values are means ⫾ SE of 5 soleus muscle samples. Values
not sharing a common letter (a, b) are significantly different (P ⬍ 0.05 by
Tukey’s multiple comparison test).
the mTOR pathway may be the reason for these variable effects
on GS, inasmuch as there is evidence that isoleucine has little
effect on mTOR signal activity, in contrast to leucine, which
results in strong activation of the pathway (1). However, it has
also been shown in rat primary hepatocytes that leucine activates mTOR signaling but does not increase GS activity (17).
Taken together, these studies suggest that there are tissue
specificities for regulation of mTOR signaling and glycogen
synthesis.
Fig. 8. Leu and Ile signaling pathway. Leu and Ile improved glucose tolerance
via upregulation of translocation of glucose transporters, whereas Leu administration increased glycogen in soleus muscle of CCl4 rats. Leu activated GS
via mammalian target of rapamycin (mTOR) signaling in a rapamycinsensitive pathway. Leu and Ile promoted glucose uptake mediated by GLUT4
translocation via novel signaling. IRS-1, insulin receptor substrate-1; cPKC,
nPKC, and aPKC, conventional, novel, and atypical PKC; PI(3,4,5)P3, phosphatidylinositol 3,4,5-trisphosphate.
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The results of our OGTTs with a 2 g/kg glucose load
suggested that the blood glucose-lowering action of leucine
was weaker than that of isoleucine (Fig. 2). It also appeared
that the differences in reduction of blood glucose levels caused
by leucine and isoleucine in the later stage of the OGTT were
greater than those in the early stage of the test. We assumed
that these differences reflected the potential of leucine and
isoleucine to activate the mTOR pathway. In a recent report,
mTOR signals were demonstrated to inhibit insulin signals by
inducing Ser/Thr phosphorylation of insulin receptor substrate-1 (32). Among the amino acids, leucine has been shown
to be the most effective activator of mTOR (1). These results
may be explained by inhibition of the endogenous insulin
signal by leucine via mTOR activation during the late stage of
the OGTT. Therefore, the early reduction in blood glucose was
caused by endogenous insulin and leucine, whereas the later
effects were caused predominantly by leucine in the absence of
insulin signaling. Inasmuch as isoleucine was a weaker activator of the mTOR signal in skeletal muscle (1), endogenous
insulin would have maintained low blood glucose levels, even
after administration of isoleucine. To explain the results shown
in Fig. 2, we need to determine the mechanism that accounts
for the greater effect on glucose tolerance of isoleucine than of
leucine.
In conclusion, this study in a rat model of cirrhosis found
that administration of leucine and isoleucine, but not valine,
decreased blood glucose levels by enhancing glucose uptake as
BRANCHED-CHAIN AMINO ACIDS IMPROVE GLUCOSE METABOLISM
ACKNOWLEDGMENTS
We are grateful to Tetsuo Kobayashi for technical assistance.
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