Eur J Nutr (2017) 56:2069–2080

DOI 10.1007/s00394-016-1245-6

ORIGINAL CONTRIBUTION

Benefits of l‑alanine or l‑arginine supplementation

against adiposity and glucose intolerance in monosodium
glutamate‑induced obesity
Thiago R. Araujo1 · Israelle N. Freitas1 · Jean F. Vettorazzi2 · Thiago M. Batista2 ·
Junia C. Santos‑Silva2 · Maria L. Bonfleur3 · Sandra L. Balbo3 ·
Antonio C. Boschero2 · Everardo M. Carneiro1 · Rosane A. Ribeiro1 

Received: 28 January 2016 / Accepted: 7 June 2016 / Published online: 17 June 2016
© Springer-Verlag Berlin Heidelberg 2016

Abstract  were smaller in Ala and Arg mice, while retroperitoneal fat
Purpose  l-alanine (Ala) and l-arginine (Arg) have been
reported to regulate pancreatic β-cell physiology and to
prevent body fat accumulation in diet-induced obesity.
Here, we assessed growth and adiposity parameters, glu-
cose tolerance, insulin secretion and the expression of insu-
lin and nutrient-regulated proteins in monosodium gluta-
mate (MSG)-obese mice supplemented with either Ala or
Arg.
Methods Male newborn C57Bl/6 mice received a daily
subcutaneous injection of MSG or saline solution (CTL
group), during the first 6 days of life. From 30 to 90 days of
age, MSG and CTL mice received or not 2.55 % Ala (CAla
or MArg groups) or 1.51 % Arg-HCl (CArg or MArg
groups) in their drinking water.
Results Adult MSG mice displayed higher adiposity
associated with lower phosphorylation of the adipogenic
enzyme, ACC, in adipose tissue. Glucose intolerance in
MSG mice was linked to lower insulin secretion and to
lower expression of IRβ in adipose tissue, as well as AS160
phosphorylation in skeletal muscle. Perigonadal fat depots
* Rosane A. Ribeiro
rosaneribeirobio@gmail.com
1
Laboratório Integrado de Morfologia, NUPEM, Universidade
Federal do Rio de Janeiro (UFRJ), Campus UFRJ‑Macaé,
Avenida São José do Barreto, 764, Macaé, RJ 27965‑045,
Brazil
2
Laboratório de Pâncreas Endócrino e Metabolismo,
Departamento de Biologia Estrutural e Funcional, Instituto de
Biologia, Universidade Estadual de Campinas (UNICAMP),
Campinas, SP, Brazil
3 oxide (NO), creatine, l-ornithine, l-glutamate and polyam-
Laboratório de Fisiologia Endócrina e Metabolismo, Centro
de Ciências Biológicas e da Saúde, Universidade Estadual do
Oeste do Paraná (UNIOESTE), Cascavel, PR, Brazil
pads were decreased by Ala supplementation only. Both
Ala and Arg improved fed-state glycemia as well as IRβ
and pAS160 content, but only Ala led to improved glucose
tolerance and insulin secretion. Adipostatic signals were
increased in MAla mice, as indicated by enhanced AMPK
phosphorylation and pACC content in fat depots.
Conclusions  Ala supplementation led to more pronounced
metabolic improvements compared to Arg, possibly due
to suppression of lipogenesis through activation of the
AMPK/ACC pathway.
Keywords  AMP-activated kinase · Insulin secretion ·
l-alanine supplementation · l-arginine supplementation ·
MSG obesity · Neuroendocrine disorder
Introduction
Although obesity predisposes to hypertension, athero-
sclerosis, stroke, some types of cancer, insulin resistance
and type 2 diabetes (T2D), efficient pharmacotherapy is
not yet available [1]. One of the reasons for this limita-
tion is the high complexity of these comorbidities, which
affect the body in a systemic fashion. For T2D treatment,
for instance, a gradual decline in β-cell adaptability to the
increasing functional demand is one factor that precipitates
the progression of clinical diabetes [2], consequently, ther-
apeutically interventions must target both insulin resistance
and endocrine pancreatic function.
l-arginine (Arg) is a natural constituent of dietary pro-
teins and was reported to regulate the synthesis of nitric
ines [3]. In addition, Arg supplementation reduces adipos-
ity and improves glucose tolerance in obese rodents and

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humans [4–7]. The mechanisms by which Arg promotes
these metabolic benefits are not yet completely understood.
One possibility is that Arg constitutes a physiological
source for NO being a substrate for NO synthase (NOS) [8,
9]. The increased NO in tissues is linked to AMP-activated
protein kinase (AMPK) activation, a key sensor and regula-
tor of cellular energy balance [10]. Some evidence suggests
that Arg regulates insulin secretion in culture experiments
[11–14]; however, its acute or long-term effects on in vivo
models remain largely unknown.
l-alanine (Ala) is a nonessential amino acid, found in
a variety of foods (meats, seafood, dairy products, seeds
and legumes), that can be biosynthesized from pyruvate
and branched chain amino acids. Ala is frequently used as
isonitrogenous control amino acid in experimental supple-
mentation approaches that investigate potential therapeutics
against obesity and its comorbidities [5, 6, 15]. However,
supplementation of high-fat diet mice with Ala acutely sup-
pressed weight gain in association with lower gene expres-
sion of fatty acid synthase in the liver, and higher gene
expression of adipose triglyceride lipase in the epididymal
fat [16]. Moreover, Ala has regulatory actions upon pan-
creatic β-cells [17–20], which have not yet been studied in
rodent models.
Neonatal administration of monosodium glutamate
(MSG) causes lesions in different hypothalamic areas,
resulting in metabolic and neuroendocrine changes that
lead to obesity [21]. MSG-obese rodents show reduced
body length, massive body fat accumulation, hyperleptine-
mia [22], hyperinsulinemia, insulin resistance and hyper-
triglyceridemia [23, 24]. Pancreatic endocrine function in
MSG obesity is adaptively modified to compensate insu-
lin resistance, with hyperplasia of β-cells [25] and insulin
hypersecretion in response to glucose [26–29]. Here, we
analyzed whether two months of Ala or Arg supplementa-
tion may regulate obesity development, glucose tolerance,
and insulin secretion and action in C57Bl/6 MSG mice.
Methods
MSG treatment and Ala and Arg supplementation
All experiments were approved by the UFRJ’s Animal
Care and Use Committee (CEUA, License No. IBQM044).
Male newborn C57Bl/6 mice received a daily subcutane-
ous injection of MSG [2 g/kg body weight (BW)/day on
the 1st and 2nd days after birth, and 4 g/kg BW/day from
3rd–6th postnatal days; MSG group] during the first 6 days
of life, or hyperosmotic saline solution (1.25 g/kg BW/day,
CTL group). From 30 to 90 days of age, the mice were ran-
domly distributed into the groups: CTL and MSG mice,
which received filtered water (CTL and MSG), or CTL and
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Eur J Nutr (2017) 56:2069–2080
MSG, which were supplemented with 2.55 % Ala (Synth,
Diadema, SP, BRA; CAla or MAla groups) or CTL and
MSG mice, which received 1.51 % Arg-HCl (Synth, Dia-
dema, SP, BRA; CArg or MArg groups) in their drinking
water [5]. The daily consumption of Ala in CAla mice was
0.124  ± 0.0009 g and for MAla, 0.125 ± 0.001 g. The
daily Arg ingested for CArg mice was 0.076 ± 0.001 g and
for MArg, 0.073 ± 0.001 g. All mice had free access to
standard rodent chow (Nuvilab, Colombo, PR, BRA) and
water; they were housed in standard cages and maintained
on a 12-h light/dark cycle (lights on 08:00–20:00 h) and
controlled temperature (21 ± 2 °C).
Intraperitoneal glucose tolerance test (ipGTT)
For ipGTT, 8-h fasted mice received an ip injection of 2 g/
kg BW glucose. Blood samples from the tip of the tail
were collected before (time 0) and at 15, 30, 60, 120 and
180 min after glucose administration for blood glucose
measurement using a glucose analyzer (Accu-Chek Per-
forma, Roche Diagnostic®, Switzerland).
Food and water intake, obesity and biochemical
parameters
During the 60 days of the supplementation period, all
groups of mice were weighted weekly. Food consumption
was measured once a week, and water intake was meas-
ured every 2 days; the results were expressed by the food or
water ingestion per day [30]. Feed efficiency was obtained
by the ratio from the total BW gained divided by the total
food consumption in the 60 days of experimental period
[30].
At 90 days of age, mice from both groups were weighed
and the nasoanal lengths were measured to obtain the
Lee index [BW (g)1/3/nasoanal length (cm) × 1000] [31].
Fasted and fed mice were euthanized by decapitation, their
blood was collected, and plasma was stored at −20 °C
for determination of insulin by radioimmunoassay (RIA).
Plasma glucose was measured in blood samples, collected
from the tip of the tail, using a glucose analyzer (as above
mentioned). Triglycerides (TG), total cholesterol (CHOL),
albumin and total proteins were measured using standard
commercial kits, according to the manufacturer’s instruc-
tions (Boehringer Mannhein®, Germany; Merck®, Ger-
many; Laborclin, Pinhais, PR, BRA).
Islet isolation and static insulin secretion
Islets were isolated by collagenase digestion of the pan-
creas. For static incubations, four islets from each mice
group were first incubated for 30 min at 37 °C in Krebs–
bicarbonate (KBR) buffer with the following composition:
Eur J Nutr (2017) 56:2069–2080 2071
115 mM NaCl, 5 mM KCl, 2.56 mM CaCl2, 1 mM MgCl2,
10 mM NaHCO3, 15 mM HEPES, supplemented with
5.6 mM glucose, 3 g of BSA/L (Sigma Chemical, St Louis,
MO, USA), and gassed with a mixture of 95 % O2/5 %
CO2, pH 7.4. This medium was discarded and replaced by
fresh KBR, and the islets were incubated for a further 1 h
with 2.8, 11.1 and 22.2 mM glucose. At the end of the incu-
bation period, the supernatant was collected and stored at
−20 °C for posterior insulin measurement by RIA using
125
I human insulin (Perkin Elmer, MA, USA). For islet
insulin content, groups of 4 islets were collected, trans-
ferred to tubes and 1 mL deionized water was added to the
islets, followed by disruption of the pancreatic cells using
a sonicator (Brinkmann Instruments, NY, USA). The islet
insulin content was also measured by RIA.
Cytoplasmic Ca2+ oscillation measurements
For intracellular Ca2+ concentration ([Ca2+]i) recordings,
islets were incubated in KBR medium containing 5.6 mM
glucose at 37° C for 2 h. During the last hour of incuba-
tion, islets were loaded with 5 µM of the Ca2+-sensitive
dye, Fura-2-acetoxymethyl esther (AM). Subsequently,
single islets were placed inside a thermostatically regulated
chamber (37° C) over poly-l-lysine-treated glass coverslips
and perifused with a BSA-free KBR buffer containing 2.8
or 11.1 mM glucose, as indicated in the figure legends.
Fura-2AM-loaded islets were imaged using an inverted
epifluorescence microscope (Nikon Eclipse TE200, Tokyo,
Japan). A ratio image was acquired every 3 s with a Cool
One camera (Photon Technology International, NJ, USA)
using a dual filter wheel equipped with 340 and 380 nm
bandpass filters, and emission was registered at 510 nm
(Photon Technology International, NJ, USA). Data were
acquired using the Image Master version 5.0 software
(Photon Technology International, NJ, USA) [32].
Western blot
After euthanasia, liver, gastrocnemius muscle and perigo-
nadal adipose tissue fragments were collected and solu-
bilized in homogenization buffer (100 mM Tris pH 7.5,
10 mM Na+ pyrophosphate, 100 mM Na+ fluoride, 10 mM
EDTA, 10 mM Na+ vanadate, 2 mM PMSF and 1 % Tri-
ton X-100) at 4 °C using an Ultra 80 II Stirrer homogenizer
(NY, USA). The extracts were then centrifuged at 15,300 g
at 4 °C for 30 min. The protein concentration in the super-
natants was assayed using the Bradford dye method [33],
using BSA as a standard curve and a commercial Brad-
ford reagent (Bio-Agency Lab., São Paulo, SP, BRA). For
SDS-PAGE, samples were diluted with loading buffer
containing β-mercaptoethanol. After heating at 95 °C
for 5 min, the proteins were separated by electrophoresis
(70 µg protein/lane in 10 % gels) and then transferred to
nitrocellulose membranes (Bio-Rad, Hercules, CA, USA).
The membranes were subsequently incubated with spe-
cific primary antibodies against the β subunit of the insu-
lin receptor (IRβ; 1:1000; cat no sc-711, Santa Cruz Bio-
Ser473
technology), phospho (p)-Akt1/2/3 (1:1000, cat. sc-7985R,
Santa Cruz Biotechnology), Akt1/2/3 (1:1000, cat. sc-8313,
Santa Cruz Biotechnology), pAMPKThr172 α (1:1000, cat.
# 2535), AMPKα (1:1000, cat # 2532), pAcetyl CoA-car-
boxylase (pACCSer79; 1:1000, cat. #3661), ACC (1:1000,
cat. #32,625), p-Akt substrate of 160 kDa (pAS160Thr642;
1:1000, cat. #4288), AS160 (1:1000, cat. #24,475) or p-hor-
mone-sensitive lipase (pHSLSer565; 1:1000, cat. #4137).
With the exceptions of IRβ, pAkt and Akt, all primary anti-
bodies were purchased from Cell Signaling Technology
(Danvers, MA, USA). Subsequently, the membranes were
incubated with secondary antibodies (1:10,000, Invitrogen,
São Paulo, SP, BRA), followed by exposure to an ECL rea-
gent. Images of specific bands were obtained and recorded
using an ImageQuant LAS 4000 Mini system (GE Health-
care Bio-Sciences, Uppsala, Sweden). The band intensi-
ties were quantified by optical densitometry using the open
source software, ImageJ (http://rsbweb.nih.gov/ij). After
assaying the target proteins, western blotting was repeated
using antibody to glyceraldehyde 3-phosphate dehydro-
genase (GAPDH; 1:10 000, cat. G9545, Sigma Chemical,
St Louis, MO, USA), as an internal control. To quantify
phosphorylation levels, densitometry of the phosphoryl-
ated bands was divided by those of the corresponding total
protein or GAPDH (as indicated in Figs. 5, 6), and subse-
quently multiplied per 100 [34].
Statistical analysis
Results are presented as mean ± SEM for the number of
determinations (n) indicated. Statistical analyses were per-
formed using one-way analysis of variance (ANOVA) fol-
lowed by Duncan’s posttest with the Statistica 7.0 software
(StatSoft, Tulsa, OK, USA), and the level of significance
was set at P < 0.05. Graphs were performed using Graph-
Pad Prism version 5.00 for Windows (GraphPad Software,
San Diego, CA, USA).
Results
Growth and obesity parameters
Body weight (BW) was measured weekly in MSG and
CTL C57Bl/6 mice, supplemented or not with Ala or Arg
for 60 days (Fig. 1a). The final BW and total BW gained
during the experimental period were similar between MSG
and CTL groups (Table 1; Fig. 1b), but body length was
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2072 Eur J Nutr (2017) 56:2069–2080

Fig. 1  Arg and Ala supplemen-

tation prevents fat deposition in
perigonadal fat pads, but only
Ala decreases retroperitoneal
fat store. Body weight (BW)
of a CTL, CAla, CArg, MSG,
MAla and MArg mice regis-
tered for 8 weeks (n = 7–10).
Mean ± SEM of the b total
BW gain during the 8 weeks
of the experimental period, c
daily food consumption, d feed
efficiency, e retroperitoneal and
f perigonadal fat pad weights
(n = 9–14). Different letters
indicate statistically significant
differences (one-way ANOVA
followed by Duncan’s posttest,
P < 0.05)

significantly lower in the MSG group (P < 0.0001). Nei-
ther Ala nor Arg supplementation altered final or total
BW gain, or body length in MAla and MArg mice, when
compared with the MSG group (Table 1; Fig. 1a, b). But,
Ala supplementation decreased daily food intake in MAla
and CAla mice, and Arg treatment reduced food con-
sumption only in MArg mice when compared with CTL
(P < 0.05; Fig. 1c). However, the feed efficiency did not
differ between the groups (Fig. 1d). In addition, MSG mice
demonstrated higher adiposity, as indicated by a 4.6- and
3.5-fold increase in retroperitoneal and perigonadal fat
stores, respectively, and a 9 % increase in the Lee index,
when compared with the CTL group (P < 0.0001; Fig. 1e,
f; Table 1). Ala supplementation significantly decreased
retroperitoneal and perigonadal fat pads by 20 % and 24 %,
respectively, when compared with MSG group (P < 0.01
and P < 0.002; Fig. 1e, f). Arg-supplemented mice showed
a 20 % reduction in perigonadal fat (P < 0.003), whereas

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Table 1  Biometric and biochemical plasma parameters in 90-day-old male CTL, CAla, CArg, MSG, MAla, Marg mice
CTL CAla CArg MSG MAla MArg

BW (g) 23 ± 1a,b 23 ± 1ª 22 ± 1a,b 22 ± 1a,b,c 21 ± 1b 20 ± 1c

Nasoanal length(cm) 9.1 ± 0.1a 9.4 ± 0.1a 9.3 ± 0.1a 8.3 ± 0.1b 8.3 ± 0.1b 8.1 ± 0.1b
Lee index 312 ± 4a 303 ± 6a 304 ± 3a 340 ± 4b 329 ± 6b 334 ± 6b
Glycemia (mM)
 Fasting 4.87 ± 0.16ª,b 4.39 ± 0.19a 4.71 ± 0.14a 5.58 ± 0.35b 4.77 ± 0.42a 5.23 ± 0.35b
 Fed 6.87 ± 0.21a 6.48 ± 0.13a 6.84 ± 0.20a 7.88 ± 0.39b 6.83 ± 0.30a 6.14 ± 0.31a
Insulin (ng/mL)
 Fasting 0.46 ± 0.06a 0.39 ± 0.11a 0.45 ± 0.07a 1.11 ± 0.17b,c 0.57 ± 0.15a,c 0.88 ± 0.17c
 Fed 0.62 ± 0.09 0.59 ± 0.11 0.59 ± 0.21 1.14 ± 0.16 0.85 ± 0.18 1.42 ± 0.36
TG (mg/dL)
 Fasting 66 ± 3 74 ± 5 74 ± 3 63 ± 5 75 ± 8 64 ± 6
 Fed 101 ± 10 106 ± 14 92 ± 11 120 ± 11 112 ± 15 101 ± 7
CHOL (mg/dL)
 Fasting 101 ± 8 87 ± 5 96 ± 5 113 ± 13 108 ± 11 120 ± 15
 Fed 101 ± 8 96 ± 7 99 ± 7 108 ± 11 111 ± 10 97 ± 8
Albumin (g/dL)
 Fasting 2.5 ± 0.1 2.4 ± 0.1 2.5 ± 0.2 2.7 ± 0.2 2.7 ± 0.2 2.8 ± 0.1
Total proteins (g/dL)
 Fasting 4.9 ± 0.2 4.5 ± 0.1 4.9 ± 0.2 4.8 ± 0.2 4.9 ± 0.2 5.0 ± 0.2

Data are mean ± SEM (n = 9–16)

Different letters indicate statistically significant differences (One-way ANOVA followed by Duncan’s posttest, P < 0.05)

retroperitoneal stores were unaltered (Fig. 1e). Finally,
no modifications in daily water intake were observed in
MSG mice (4.8 ± 0.09 mL), in comparison with CTL
(4.9  ± 0.02 mL). Ala and Arg treatments did not altered
this parameter in controls (5.0 ± 0.06 and 5.1 ± 0.09,
respectively) and MSG-supplemented (4.9 ± 0.05 and
4.9 ± 0.07) mice.
Glucose homeostasis
MSG-treated mice presented higher glycemia in the
fed state and in fasting hyperinsulinemia (P < 0.04 and
P < 0.001; Table 1). Both Ala and Arg supplementations
normalized fed glycemia in MSG mice. Fasting plasma
insulin was partially reduced in Ala-supplemented MSG
mice (Table 1). In addition, plasma lipids and circulating
proteins were not altered by either MSG or amino acid sup-
plementation (Table 1).
During the ipGTT, maximal blood glucose was observed
at 30 min in all groups (Fig. 2a). In MSG mice hypergly-
cemia was observed at 15 min, persisting during the entire
test (P < 0.001; Fig. 2a). Ala supplementation reduced
blood glucose at 60 min in MAla mice, when compared
with MSG (P < 0.04; Fig. 2a). Total glycemia, indicated
by the AUC, was also higher in MSG compared with CTL
group (P < 0.0001; Fig. 2b). Total blood glucose, during
ipGTT, was decreased by 21 % in MAla mice, compared
with MSG (P < 0.0001; Fig. 2b). Blood glucose, over time,
was not significantly changed by Arg treatment, and total
glycemia was only partially reduced in the MArg group
(Fig. 2b).
Insulin secretion
Glucose tolerance is mainly determined by the insulin
secretion capacity of the pancreatic β-cells. Insulin granule
exocytosis is triggered by increased Ca2+ influx through
voltage-gated Ca2+ channels, which are opened in response
to membrane depolarization, as a consequence of glucose
metabolism [35].To determine whether glucose intolerance
in MSG was linked to Ca2+ influx-related β-cell dysfunc-
tion and if it could be improved by amino acid supplemen-
tation, we assessed insulin secretion and Ca2+ handling
in response to increasing glucose concentrations in islets
isolated from MSG and CTL mice supplemented, or not,
with Ala or Arg. Islets isolated from MSG mice secreted
less insulin in response to 11.1 and 22.2 mM glucose, when
compared with CTL islets (P < 0.0001 and P < 0.03, respec-
tively; Fig. 3). This lower secretion was associated with a
reduced amplitude of Ca2+ influx in response to 11.1 mM
glucose in MSG islets, compared with CTL (P < 0.02;
Fig.  4d, a, h, respectively), whereas total Ca2+ entry and
oscillatory frequency remained unaltered (Fig. 4g, i). Insu-
lin release at 11.1 and 22.2 mM glucose from MAla islets

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Fig. 2  Ala supplementation, but not Arg, improves glucose toler-
ance in MSG mice. a Changes in blood glucose during the ipGTT
in 90-day-old CTL, CAla, CArg, MSG, MAla and MArg mice
(n  = 7–14). b Mean ± SEM of the total plasma glucose concentra-
tions during the ipGTT, expressed by the AUC. $MSG is different
Fig. 3  Ala supplementation normalized glucose-induced insulin in
MSG obesity. Glucose-induced insulin secretion in islets isolated
from 90-day-old MSG and CTL mice supplemented or not with Ala
or Arg. Groups of 4 islets were incubated for 1 h with different glu-
cose (G) concentrations. Each bar represents mean ± SEM of 6–7
independent experiments. Different letters over the bars indicate sta-
tistical differences between the groups for the same glucose concen-
tration evaluated (one-way ANOVA followed by Duncan’s posttest,
P < 0.05)
was fully rescued to CTL levels (Fig. 3). This effect was,
at least partly, due to the normalization of the amplitude of
the Ca2+ influx (Fig. 4e, h). However, in MArg islets, both
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Eur J Nutr (2017) 56:2069–2080
from CTL, CAla and CArg groups (P < 0.05); * MSG, MAla and
MArg groups are different from CTL (P < 0.05); # MSG and MArg
mice were different from CTL (P < 0.05). Different letters over the
bars indicate statistically significant differences (one-way ANOVA
followed by Duncan’s posttest, P < 0.05)
insulin secretion and Ca2+ influx amplitude were only par-
tially restored to levels of between those of the MSG and
CTL groups (Figs. 3, 4h). Ala treatment also enhanced the
total and amplitude of [Ca2+]i in CAla islets in response
to 11.1 mM glucose, when compared with CTL (P < 0.01
and P < 0.05; Fig. 4b, g, h). Furthermore, no alteration in
islet total insulin content was observed in MSG (57 ± 3 ng/
islet), when compared with CTL islets (66 ± 4 ng/islet).
Ala or Arg supplementation did not alter islet insulin con-
tent in MSG (MAla 53 ± 5 and MArg 66 ± 6 ng/islet) or
control groups (CAla 56 ± 5 and CArg 66 ± 6 ng/islet).
Thus, impairment of β-cell function in MSG islets was due
to a defective secretory mechanism involving lower Ca2+
influx, restored by Ala but not Arg supplementation.
Expression of proteins involved in insulin signaling
and the AMPK pathway in peripheral tissues
To determine whether glucose intolerance in MSG mice
was also associated with a peripheral component of insu-
lin resistance, we assessed the expression of the β subunit
of the insulin receptor (IRβ) as well as the basal phospho-
rylation of protein kinase B/Akt (p-Akt), one of its down-
stream effectors. Expression of IRβ in perigonadal adipose
tissue was significantly reduced when compared with CTL
mice (P < 0.001; Fig. 5e). Hepatic and muscle IRβ protein
expression also displayed a trend toward a reduction in the
MSG group (Fig. 5a, c). The basal state p-Akt remained
unaltered in all investigated tissues (Fig. 5b, d, f).
Furthermore, we analyzed components of the AMPK
pathway, which is associated with insulin sensitizing and
Eur J Nutr (2017) 56:2069–2080 2075
Fig. 4  Representative curves of glucose-induced cytoplasmic
Ca2+ oscillations in islets isolated from 90-day-old CTL (a), CAla
(b), CArg (c), MSG (d), MAla (e) and MArg (f) mice. Data are the
ratio of F340/F380 registered for each group. g The area under the
curve (AUC), h amplitude and i frequency of Ca2+ oscillations of
the [Ca2+]i in response to 11.1 mM glucose. The experiments were
blood glucose lowering effects [10]. Even though MSG
mice showed only a tendency toward lower pAMPK/
AMPK ratios in the liver, muscle and adipose tissue
(Fig.  6a–c), some of their downstream targets, such as
pAS160 in muscle, and pACC and pHSL in adipose tissue,
were significantly reduced when compared with the CTL
group (P < 0.001, P < 0.0001 and P < 0.002, respectively
(Fig. 6d, f, g).
Ala and Arg treatments normalized IRβ protein expres-
sion in adipose tissue from MAla and MArg mice (Fig. 5e).
Ala-supplemented MSG mice showed enhanced p-AMPK/
AMPK in all tissues measured (P < 0.0001; Fig. 6a, c, e).
This effect was accompanied by normalization in pAS160
content in muscle, and pACC and pHSL in adipose tissue
(Fig. 6d, f, g). No difference in hepatic pACC protein con-
tent was observed between the groups (Fig. 6b). Arg sup-
plementation had a minor impact upon the AMPK pathway,
performed in a perifusion system in a medium that contained 2.8 or
11.1 mM glucose (G2.8 and G11.1, respectively). Data are reported
as mean ± SEM that were obtained from 5 to 10 independent experi-
ments. Different letters represent statistically significant differences
(one-way ANOVA followed by Duncan’s posttest, P < 0.05)
as indicated by normalization of only pAS160 content in
muscle (Fig. 6d).
Discussion
MSG-induced hypothalamic lesions after birth in rodents
are a model largely employed to better understand the
pathophysiology of morbid obesity. In our study, we
used C57Bl/6 mice, a mouse strain widely used in meta-
bolic studies; however, data on MSG-induced metabolic
damages in this background are scarce. Previous reports
showed that although adult C57Bl/6 MSG mice had mas-
sive body fat deposition and hyperleptinemia, they did not
develop hyperinsulinemia or insulin resistance [22] fea-
tures that were observed in different strains of rats [23, 25,
29, 36–38] and albino mice [28, 39, 40] treated with MSG.
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2076
Fig. 5  Ala and Arg supplementation normalizes IRβ protein con-
tent in adipose tissue in MSG-treated mice. IRβ and pAkt/Akt pro-
tein expressions in the liver, gastrocnemius muscle and perigonadal
adipose tissue from 90-day-old CTL, CAla, CArg, MSG, MAla
and MArg mice (n  = 4–8). Fragments were obtained from the liver
(a and b), skeletal muscle (c and d) and white adipose tissue (e and
f) and used for immunoblotting experiments. The bars represent
mean ± SEM of densitometric values. Different letters represent sta-
tistically significant differences (one-way ANOVA followed by Dun-
can’s posttest, P < 0.05)
13
Eur J Nutr (2017) 56:2069–2080
Fig. 6  Ala and Arg normalize basal AS160 inhibition, but only Ala ▸
supplementation promotes normalization of ACC and HSL phos-
phorylation in MSG obesity. Phospho-AMPK/AMPK, pACC/ACC,
pAS160/AS160 and pHSL protein expressions in the liver (a and b),
gastrocnemius muscle (c and d) and perigonadal adipose tissue (e,
f and g) from 90-day-old MSG and CTL mice supplemented or not
with Ala or Arg. Protein extracts were processed for Western blot.
Bars represent the mean ± SEM of the values, determined by optical
densitometry (n = 4–8 mice). Different letters over the bars represent
statistically significant differences (one-way ANOVA followed by
Duncan’s posttest, P < 0.05)
Here, we provide evidence that C57Bl/6 MSG mice pre-
sented hyperglycemia along with critical features of early
onset T2D, such as hyperinsulinemia, glucose intolerance
associated with lower glucose-induced insulin secretion,
peripheral down-regulation of the IR and impaired AMPK
downstream signaling.
The benefits of Arg supplementation against the devel-
opment of obesity were demonstrated in genetically [5] and
diet-induced obesity [6]. Arg treatment also ameliorated
glucose tolerance in high-fat diet rats [6] and in hyper-
lipidemic hamsters [4]. This amino acid also decreases the
probability of becoming glucose intolerant and the inci-
dence of diabetes in subjects supplemented for 14 months
with Arg [7].
However, the anti-obesity actions of Ala have not
yet been reported. In fact, this amino acid has been fre-
quently used as an isonitrogenous control in studies that
have investigated the effects of amino acid supplemen-
tation on obesity and metabolic disturbances [5, 6, 15].
Here, we verified that Ala and Arg supplementation effi-
ciently prevented fat deposition in MSG mice, but Ala
supplementation was more effective since the amino
acid decreased the two abdominal fat stores evaluated.
In addition to the prevention of obesity, Ala also ame-
liorates glucose tolerance in MAla mice. All these find-
ings indicate that, like Arg, Ala can also improve body
glucose control and lipid metabolism in obesity. The dis-
crepancy between our results and the above mentioned
reports that use Arg supplementation may be due to the
rodent strain used and time employed for beginning the
treatment.
One of the mechanisms that probably accounted for glu-
cose intolerance in MSG mice, in our study, was the lower
insulin secretory capacity in response to glucose. Impor-
tantly, compensatory islet hyperfunction was reported
in MSG Wistar rats [25, 27, 29] and MSG albino mice
[26, 28]. Thus, our data indicate that the C57Bl/6 strain
was more susceptible to the disruption of β-cell function,
induced by MSG, which can contribute to the early onset
of T2D.
Eur J Nutr (2017) 56:2069–2080 2077

13

2078
Arg is known to regulate endocrine pancreatic functions.
This cationic amino acid enters the β-cell via the electro-
genic transporter mCAT2A, causing membrane depolariza-
tion and increased [Ca2+]i [11]. Recently, Arg was reported
to enhance Ca2+ influx in β-cells through regulation of the
αi2 subunit of the G proteins [13]. The amino acid dose-
dependently prevented cytokine-induced β-cell apoptosis
due to its conversion to l-glutamate, which enhances anti-
oxidant defenses [12]. In contrast, incubation of rat islets
with 1.5 or 10 mM Arg for 48 h decreased insulin secre-
tion, reduced islet-cell proliferation and increased apopto-
sis through activation of the endoplasmic reticulum stress
response [14]. Possibly, these dose-dependent effects of
Arg may contribute to an incomplete preservation of the
islet function in the MArg group.
The insulinotropic action of Ala has been related to its
co-transport with Na+, which results in β-cell depolariza-
tion and increased [Ca2+]i [17]. In the β-cell line, BRIN-
BD11, Ala was shown to be metabolized and increase
glutamate and aspartate levels. Furthermore, incubation of
BRIN-BD11 cells with 10 mM Ala increases glucose utili-
zation and insulin secretion [19]. Incubation of BRIN-BD11
cells with 10 mM Ala for 24 h enhances the expression of
genes involved in cellular signaling, metabolism, protein
synthesis, apoptosis and the cellular stress response [20].
As such, normalization of insulin release in MAla islets
is probably associated with the preservation of glucose-
induced β-cell depolarization and [Ca2+]i in response to
glucose, since the amplitude of the Ca2+ influx was nor-
malized in MAla islets. Additional effects of the amino acid
upon gene expression, not addressed in our study, may also
be considered.
The reduction in IRβ protein content in the adipose tis-
sue of MSG mice may have decreased the action of insu-
lin and impair glucose tolerance in this group. The down-
regulation of the IRβ protein may be associated with basal
hyperinsulinemia in MSG group, working in a negative
feedback mechanism whereby insulin dose- and time-
dependent desensitizes its target tissues [41, 42]. A previ-
ous study in 10-week-old MSG rats, however, showed that
reductions in insulin-induced IR phosphorylation in periph-
eral tissues were not linked to any alteration in its total
protein content [23]. Our results indicate again that MSG-
induced metabolic changes differ depending on the rodent
strain used.
The AMPK pathway is known to promote effects in
peripheral tissues to reduce the risk for obesity and insu-
lin resistance. AMPK phosphorylates ACC, which inhibits
this enzyme, decreasing the conversion of acetyl-CoA to
malonyl-CoA. This effect activates carnitine palmitoyl-
CoA transferase-1, enhancing fatty acid oxidation [10]. In
adipocytes, AMPK also has an antilipolytic action medi-
ated by the phosphorylation of HSL at the Ser 565 residue,
13
Eur J Nutr (2017) 56:2069–2080
decreasing its activation by protein kinase A [43]. In addi-
tion, AMPK mediates glucose transport in resting skeletal
muscle via phosphorylation and inhibition of AS160, which
increases GLUT-4 translocation to the membrane [44].
Here, MSG mice showed impaired AMPK downstream
signaling, as indicated by lower pACC, pHSL and pAS160
content in peripheral tissues. This effect contributes to
increased fat deposition in perigonadal fat stores of MSG
group, possibly due to increased lipogenesis. The reduction
in HSL phosphorylation, mediated by AMPK in adipose
tissue, may increase lipolysis when fatty acids demand is
low, which may aggravate the metabolic status of MSG
mice. The lower basal pAS160 protein expression in skel-
etal muscle is an additional mechanism linked to impaired
glucose control after MSG treatment.
Although the mechanisms through which Arg modu-
lates glucose homeostasis and lipid metabolism regulation
are not completely understood, there is much evidence
to indicate that NO, generated from Arg and its products,
can activate AMPK [8, 9]. However, no effect of Ala upon
AMPK activation has been demonstrated to date. Here,
we observed a significant increase in the expression of
pAMPK/AMPK protein in all peripheral tissues analyzed
in MAla mice. While basal AMPK activation only showed
a tendency toward an increase in MArg peripheral tissues,
this effect may have been associated with long-term expo-
sure to Arg. It has been shown that short-term incubation
of human umbilical vein endothelial cells (HUVEC) with
30 µM Arg enhanced AMPK activity, NO synthesis and
cellular glucose uptake. In contrast, HUVEC incubated
for 7 days with 30 µM Arg, displayed a decrease in AMPK
action together with reductions in endothelial NOS action
and NO levels, but augmented superoxide and peroxynitrite
(ONOO−) cellular content [9]. Conversely, when we inves-
tigated the inhibition of AS160, a downstream target of
AMPK’s, both MArg and MAla skeletal muscles presented
similar improvements in pAS160 muscle content, which
possibly contributes to improve fed glycemia in MArg
and glucose tolerance in MAla mice. In contrast, AMPK’s
downstream target proteins, ACC and HSL, were more
phosphorylated only in MAla perigonadal adipose tissue,
which may have prevented fat deposition in this group.
Furthermore, the improved action of AMPK may have pre-
vented IRβ down-regulation in the adipose tissue of MAla
and MArg mice, since previous reports have shown that
metformin prevents insulin-induced decreases in IR biding
sites in adipocytes [42].
In summary, C57Bl/6 mice, treated with MSG, devel-
oped obesity and glucose intolerance in association with
lower islet secretory function and down-regulation of IRβ
in adipose tissue. Increased fat deposition in MSG mice is
due, at least in part, to decreased inhibition of ACC in adi-
pose tissue, which may enhance lipogenesis. For the first
Eur J Nutr (2017) 56:2069–2080 2079
time, we demonstrate that Ala supplementation prevents
fat deposition in perigonadal adipose tissue by enhanc-
ing AMPK-induced inhibition of ACC. Ala also improved
glucose tolerance in MSG obesity by maintaining the
islet secretory function, IRβ expression in adipose tissue,
together with a better pAS160 protein expression in skel-
etal muscle. Arg also had some benefits against obesity and
glucose homeostasis in MSG mice, but all of these Arg-
mediated effects were less pronounced than those promoted
by Ala supplementation.
Acknowledgments  This study was supported by Grants from Con-
selho Nacional para o Desenvolvimento Científico e Tecnológico
(CNPq 442846/2014-2), Fundação Carlos Chagas Filho de Amparo à
Pesquisa do Estado do Rio de Janeiro (Faperj E-26/110.921/2013 and
E-26/101.129/2014) and Fundação de Amparo à Pesquisa do Estado
de São Paulo (Fapesp). We are grateful to Gilliana Neves and Josué
Almeida from Biotério de Experimentação de Roedores for animal
care and Nicola Conran for editing the English.
Compliance with ethical standards  Saad MJ (2007) L-glutamine supplementation induces insulin
Conflict of interest  The authors have no conflict of interest.
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