nutrients

Article
Oral Glutamine Supplementation Reduces Obesity,
Pro-Inflammatory Markers, and Improves Insulin
Sensitivity in DIO Wistar Rats and Reduces Waist
Circumference in Overweight and Obese Humans
Kahlile Youssef Abboud 1,† , Sabrina Karen Reis 1,† , Maria Eduarda Martelli 1,† ,
Olivia Pizetta Zordão 2,† , Fabiana Tannihão 1 , Alessandra Zanin Zambom de Souza 1 ,
Heloisa Balan Assalin 2 , Dioze Guadagnini 2 , Guilherme Zweig Rocha 2 ,
Mario Jose Abdalla Saad 2 and Patricia Oliveira Prada 1,2, *
1 School of Applied Sciences, State University of Campinas (UNICAMP), Limeira 13484-350 SP, Brazil;
[email protected] (K.Y.A.); [email protected] (S.K.R.);
[email protected] (M.E.M.); [email protected] (F.T.);
[email protected] (A.Z.Z.d.S.)
2 Department of Internal Medicine, State University of Campinas (UNICAMP), Campinas 13083-887 SP, Brazil;
[email protected] (O.P.Z.); [email protected] (H.B.A.); [email protected] (D.G.);
[email protected] (G.Z.R.); [email protected] (M.J.A.S.)
* Correspondence: [email protected] or [email protected]; Tel./Fax: +55-19-35218950
† Equal contributors.




Received: 1 February 2019; Accepted: 24 February 2019; Published: 1 March 2019


Abstract: In the present study, we aimed to investigate whether chronic oral glutamine (Gln)
supplementation may alter metabolic parameters and the inflammatory profile in overweight and
obese humans as well as whether Gln may modulate molecular pathways in key tissues linked
to the insulin action in rats. Thirty-nine overweight/obese volunteers received 30 g of Gln or
alanine (Ala-control) for 14 days. Body weight (BW), waist circumference (WC), hormones, and
pro-inflammatory markers were evaluated. To investigate molecular mechanisms, Gln or Ala was
given to Wistar rats on a high-fat diet (HFD), and metabolic parameters, euglycemic hyperinsulinemic
clamp with tracers, and Western blot were done. Gln reduced WC and serum lipopolysaccharide (LPS)
in overweight volunteers. In the obese group, Gln diminished WC and serum insulin. There was a
positive correlation between the reduction on WC and LPS. In rats on HFD, Gln reduced adiposity,
improved insulin action and signaling, and reversed both defects in glucose metabolism in the liver
and muscle. Gln supplementation increased muscle glucose uptake and reversed the increased
hepatic glucose production, in parallel with a reduced glucose uptake in adipose tissue. This insulin
resistance in AT was accompanied by enhanced IRS1 O-linked-glycosamine association in this tissue,
but not in the liver and muscle. These data suggest that Gln supplementation leads to insulin
resistance specifically in adipose tissue via the hexosamine pathway and reduces adipose mass,
which is associated with improvement in the systemic insulin action. Thus, further investigation
with Gln supplementation should be performed for longer periods in humans before prescribing as a
beneficial therapeutic approach for individuals who are overweight and obese.

Keywords: obesity; glutamine supplementation; inflammation; insulin sensitivity; cytokines; clamp;

LPS; hexosamine

Nutrients 2019, 11, 536; doi:10.3390/nu11030536 www.mdpi.com/journal/nutrients

Nutrients 2019, 11, 536 2 of 16

1. Introduction
Obesity has become a significant public health problem worldwide, and it is associated with
various comorbidities [1–5]. Obesity is considered a low-grade inflammatory disease and the degree
of inflammatory status correlates positively with the development of insulin resistance and type 2
diabetes mellitus [6–8]. The white adipose tissue has a primary role sensing and managing energy
homeostasis [7]. Consistently, human and rodents studies demonstrated that, under a positive
energy balance, the white adipose tissue triggers an immune response, which develops low-grade
inflammation milieu, associated with infiltration of immune cells [7–9]. Additionally, products from
intestinal microbiota as lipopolysaccharides (LPS) can also contribute to this inflammatory state in
obesity [10–12].
Many efforts have been made to prevent and treat obesity and to reduce the low-grade
inflammatory status, including hypocaloric diets combined or not combined with physical activity
and drugs in humans [13]. However, the complexity and time spent in this treatment often lead to
weight recovery [14]. Thus, it will be useful if an obesity treatment strategy takes into account single
nutrient supplementation. Diets with glutamine (Gln) supplementation have aroused interest since
they can mitigate the release of cytokines, reduce organ damage, and improve survival of mice and
humans with endotoxemia [15–20]. However, studies that evaluated the potential of oral glutamine
supplementation decreasing body weight and fat mass in an obese human being are scarce [21–23].
Chronic oral glutamine supplementation was shown to improve fasting blood glucose and A1c as
well as reduced body fat and waist circumference (WC) in type 2 diabetic individuals [23]. In another
study, oral glutamine supplementation for four weeks was able to reduce body weight (BW) and WC,
but not insulinemia and HOMA-IR in overweight and obese female patients [22]. However, none of
these studies investigated possible pathophysiological mechanisms of how glutamine supplementation
could contribute to weight and adipose mass reduction and improve metabolic parameters.
Insulin has a potent lipogenic effect on adipose tissue [7]. This effect is related to the tyrosine
phosphorylation of the insulin receptor by insulin, which induces PI3K/Akt pathway activation
that leads to glucose transport, and lipogenesis afterwards [7,8]. Mice with specific insulin receptor
disruption on fat (FIRKO mice) are protected from obesity induced by a high fat diet [24]. On the
other hand, animals on the high fat diet usually displayed insulin resistance in the liver and muscle
but not in the adipose tissue [25,26]. These data argue that, in some specific conditions, the lack of
insulin effects in adipose tissue may protect from obesity and some of its comorbidities. Previous cell
culture studies suggested that glutamine was able to reduce insulin action in adipocytes, but not on L6
muscle cells [27–29]. Accordingly, the lack of effect of insulin on the adipose tissue through glutamine
supplementation might be beneficial in reducing lipogenesis and, hence, fat accumulation in vivo.
Thus, we combined human and animal models to deeper understand the mechanisms by which
glutamine may reduce obesity and its comorbidities. Herein, we aimed to investigate (1) whether
chronic oral Gln supplementation may alter anthropometric, metabolic parameters and also the
inflammatory profile in overweight and obese humans in a proof of concept study. We also attempted
to investigate (2) whether chronic Gln supplementation via gavage may alter these same parameters in
high-fat diet (HFD) rats, which are integrated with an investigation of insulin action and signaling in
specific tissues as liver, muscle, and adipose tissue. We attempted to understand, at a molecular level,
this beneficial effect of glutamine.

2. Materials and Methods

2.1. Human Study

This study was conducted by the Declaration of Helsinki (1964) and was approved by the Ethics
Committee of the Department of Internal Medicine at State University of Campinas (UNICAMP),
Campinas, SP, Brazil. All the subjects gave written informed consent.
Nutrients 2019, 11, 536 3 of 16

All volunteers were employees with a majority of nurses at the Sumare State Hospital in Sumare
city, São Paulo state, Brazil. A total of 150 volunteers were randomly recruited via advertisements
placed around the hospital. In this double-blind study, 39 volunteers completed the intervention
period. The inclusion criteria to participate were as follows: men or women adults aged between
20 and 60 years old and diagnosed as overweight or obese. Subjects who reported renal or thyroid
disease, pregnancy, taking an antidepressant, and anorectic or laxative drugs were excluded from
the study. Before beginning the study, body weight and height were measured using a Filizola scale
with a calibrated digital scale and stadiometer, respectively (PL 200 model). The Body Mass Index
(BMI) (kg/m2 ) was calculated as weight (kg) divided by height (m2 ) squared. Only overweight
(BMI ≥ 25 kg/m2 ) and obese (BMI ≥ 30 kg/m2 ) volunteers were included in the study (WHO, 2000).
On the first day, volunteers were random divided into four groups according to the BMI and the
supplement that they would receive: Overweight Alanine (Ala), Obese Ala, Overweight Glutamine,
and Obese Gln. Afterward, blood samples were collected and waist circumference was measured.
The volunteers received a kit containing small packs with 15 g of amino acid (Gln or Ala) each.
They were instructed to take two packs per day, taking a total of 30 g of amino acids per day.
The supplementation lasted for 14 days. The volunteers were instructed to mix the pack content
in a cup of water (200 mL) before drinking and maintain the same levels of physical activity and the
same diet during the 14 days of supplementation. The second measurement was done 15 days after
the supplementation started. Fasting volunteers came to the hospital for the second time for BW and
WC measurements and blood sample collections.
Biochemical Analysis was conducted by blood samples, which were obtained before and after the
supplementation from the same volunteer. Overnight fasted volunteers had blood samples collected
into tubes placed on ice. After collection, blood samples were immediately centrifuged at 1500 rpm
for 15 min at 18 ◦ C using a Centrifuge Biofuge Stratos (Hereaus, Dijkstra Vereenigde, Lelystad,
Netherlands). The serum obtained was separated and transferred into 2 mL Eppendorf and stored
at −80 ◦ C until analysis. Glucose concentration was determined using the Glucose Liquiform Test
(Labtest, Brazil) that applied the glucose oxidase method. All the other assays were quantified by the
specific commercial enzyme-linked immunosorbent assay (ELISA). The human insulin (EZHI-14K)
kit was from Millipore® , St. Charles, Missouri, United States. Human TNF-α (DTA00C), human
IL-1β (DLB50), and human IL-6 (D6050) kits were from R&D Systems Inc., Minneapolis, MN, USA.
To determine serum lipopolysaccharides (LPS) levels, the Limulus Amebocyte Assay from Cambrex
(LAL kit endpoint-QCL-1000, Lonza, Walkersville, MD, United States) was used. Analyses were
performed by following the specific instructions for each manufactory. HPLC system/SCL-10avp
(Shimadzu Scientific Instruments, Columbia, MD, USA) and the CLASS-VP 6.12 software Class VP
were used to measure serum glutamine and alanine [30] levels in order to assess the adherence to
glutamine or alanine supplementation. The serum was collected before and after supplementation.
To assess the caloric intake of the individuals, we applied a 24-h food record before and after
the supplementation. For analysis of the 24-h food record, we used the software Diet Pro 4.0 (Viçosa,
Brazil). To assess physical activity, subjects were informed of their physical activity before and after
supplementation during an interview.

2.2. Animal Study

Male Wistar rats received a chow diet from weaning until eleven-weeks-old. Then the rats were
divided into two groups with similar body weight as follows: 1) rats that will continue receiving
chow diet (chow) with 70% calories from carbohydrate, 20% from proteins, and 10% from fat, and
2) rats that start to receive a high-fat diet with 55% calories from fat, 29% from carbohydrates, and
16% from proteins. After four weeks on these diets, the rats fed with a high-fat diet were divided into
two other groups. One group received glutamine supplementation (HF+Gln) and the second group
received alanine supplementation (HF+Ala) for four more weeks. All diets and water were ad libitum
offered. Both supplements (alanine and glutamine) were given in the drinking water (35 mL), and
Nutrients 2019, 11, 536 4 of 16

were prepared and replaced every day (4%). In addition, a gavage was done with alanine or glutamine
(0.4 g in 1 mL) three days a week for four weeks. In accordance with Reagan-Shaw et al. [31], the ratio
of conversion from human to rat studies is around six. In this regard, we used a dose of ~0.4 g/kg of
glutamine in humans and ~2.4 g/kg in rats.
Body weight was followed for eight weeks. At the end of the study, the epididymal,
retroperitoneal, and mesenteric fat pads were weighed.
Food was withdrawn 12 h before the blood samples collection, and the serum glucose, insulin,
TNF-α, and IL-6 levels were determined by ELISA using specific kits from Linco (St Charles, MO, USA;
Pierce, Rockford, IL, USA). Serum glutamine levels were determined as described before [30].
The measurement of oxygen consumption/carbon dioxide production and the respiratory
exchange ratio (RER) were completed in fed rats by an indirect open circuit calorimeter (Oxymax
Deluxe System, Columbus Instruments, Columbus, OH, USA), as previously described [32].
Rats were deeply anesthetized, and, as soon as the loss of pedal and corneal reflexes, the abdominal
cavity was opened, and saline (200 µL) or insulin (2 µg in 200 µL) were injected in the exposed portal
vein. A fragment of liver was removed after 30 s, and gastrocnemius and the epididymal fat pad
were removed after 90 s. Fragments of tissues were homogenized immediately in extraction buffer,
as described elsewhere [25]. The samples were then centrifuged at 15,000 rpm and 4 C for 40 min
to remove insoluble material, and the supernatants were used for immunoblotting, as previously
described [25,33,34] by using the following antibodies: O-linked N-acetylglucosamine, (MA1-072
from Affinity Biologicals, Ancaster, Ontario, Canada), Insulin Receptor Substrate 1 (IRS-1) (C-20)
(sc-559), and phosphorylated protein kinase B (p-AKT-1/2/3) (Ser 473) (sc-7985-R) from Santa Cruz
Biotechnologies (Dallas, TX, USA).
The hyperinsulinaemic-euglycemic clamp was performed using a previous protocol [25].
In summary, after five hours of fasting, rats were anesthetized, and two catheters were inserted
including one into the left jugular vein (tracer infusion) and the other into the carotid artery (blood
samples). The hyperinsulinemic-euglycemic clamp study was conducted with a continuous insulin
infusion up to 120 min (rate of 3.6 mU/kg body weight per minute) to raise the concentration of
insulin in plasma for approximately 800–900 pmol/L. Every 5 min, the blood samples were collected
for measuring blood glucose and 10% unlabeled glucose at variable rates was infused, to maintain the
glucose concentration at fasting levels. To estimate the insulin-stimulated whole-body glucose flux,
a prime continuous HPLC-purified [3-3 H] glucose (10 µCi boluses, 0.1 µCi/min) was also infused.
A bolus (10 µCi) of 2-[14 C]DG1 was infused at 90 min of the clamp to estimate skeletal muscle
and visceral fat insulin-stimulated glucose-transport activity and metabolism. Blood glucose was
determined at 80, 90, 100, 110, and 120 min after the clamp procedure starts for plasma [3 H] glucose and
2-[14 C]DG1 concentration measurements afterwards. At the end of 120 min, all rats were euthanized
by anesthesia i.v. injection. The gastrocnemius skeletal muscles from hind limbs and the epididymal
fat pad were taken within 2 min. All tissues were weighted, frozen with liquid nitrogen, and stored at
−80 ◦ C for future analysis.

Determination of the Nuclear Factor Kappa B (NF-κB) Activation

NF-κB p50 activation was determined in nuclear extracts from muscle and adipose tissue by
ELISA (89858, Thermo Fisher Scientific Inc., Rockford, IL, USA), according to the recommendations of
the manufacturer.

2.3. Statistical Analysis

The human data were collected before and after Gln and Ala supplementations and were analyzed
in separated subgroups, according to BMI (overweight and obese) by using the Student’s t test paired
two-tailed for parameters with a normal distribution or the Wilcoxon test paired two-tailed for
parameters with a nonparametric distribution. A correlation between WC and LPS was made by using
the Pearson test.
Nutrients 2019, 11, 536 5 of 16

The animal study included three different groups, which were analyzed by One-Way ANOVA
with the Bonferroni post-test. In both human and animal studies, the level of significance adopted was
p < 0.05.

3. Results

3.1. Glutamine Supplementation in Overweight and Obese Subjects Reduce Waist Circumference and
Circulating LPS
Thirty-nine adult volunteers who are overweight or obese (3 men and 36 women) were included
and completed in the study protocol. They were 38.3 ± 7.2 years old. The only volunteers used
in the study were the ones who maintained similar physical activity. The caloric intake of the
individuals was recorded for 24 h, and only the volunteers that maintained similar caloric intake
were used in the study. Volunteers did not display differences in caloric intake before and after
supplementations (Overweight volunteers supplemented with Ala Before 2298 kcal/day ± 542.1
n = 8. After 2343 kcal/day ± 327.3 n = 8. Obese volunteers were supplemented with Ala before
1552 kcal/day ± 490.7 n = 7. After 1545 kcal/day ± 317.9 n = 7. Overweight volunteers were
supplemented with Gln before 2069 kcal/day ± 1281 n = 11 and after 1923 kcal/day ± 624.7 n = 11.
Obese volunteers were supplemented with Gln before 1816 kcal/day ± 470.6 n = 13 and after
2025 kcal/day ± 567.5 n = 13).
Data from overweight volunteers are presented in Table 1 and Table S1. Data from obese
volunteers are presented in Table 2 and Table S2. No differences were observed in the body weight
and BMI after supplementation with Gln or Ala in overweight volunteers (Table 1). However, the
glutamine supplementation induced a significant decrease in the waist circumference of overweight
subjects (Table 1). Serum glucose, insulin levels, IL-1β, IL-6, and TNF-α levels were not different
after glutamine or alanine supplementation (Table 1). However, the supplementation with glutamine,
but not with alanine, reduced circulating LPS levels in overweight subjects (Table 1). In addition,
Ala levels were increased in Ala supplemented volunteers and Gln levels were also increased in Gln
supplemented volunteers (Overweight volunteers supplemented with Ala Before 448.09 µmol/L ± 52.3
n = 8. After 554.46 µmol/L ± 57.64 n = 8. Overweight volunteers supplemented with Gln Before
419.88 µmol/L ± 35.11 n = 11. After 531.09 µmol/L ± 36.52 n = 11 and Tables S1 and S2).

Table 1. Differences in metabolic characteristics of overweight volunteers before and after

supplementation with glutamine or alanine.

Overweight Volunteers
Ala Suppl Difference Gln Supplemental
between After and Before p-Value Difference between After p-Value
(n = 8) and Before (n = 11)
Height (m)
Weight (kg) 0.4 ± 0.74 0.17 −0.44 ± 0.89 0.13
BMI (kg/m2 ) 0.16 ± 0.29 0.16 −0.17 ± 0.32 0.10
Serum amino acid levels (µmol/L) 106.36 ± 12.55 0.0001 111.20 ± 13.83 0.0001
WC (cm) −0.19 ± 0.8 0.52 −1.82 ± 1.4 0.001 *
Glucose (mmol/L) −0.08 ± 0.21 0.34 0.06 ±0.6 0.83
Insulin (µU/mL) 0.16 ± 0.74 0.33 −1.00 ± 2.89 0.41
TNF-α (pg/mL) 0.04 ± 0.96 0.91 −2.88 ± 3.94 0.28
IL-1β (pg/mL) 0.01 ± 0.17 0.84 −0.14 ± 0.33 0.31
IL-6 (pg/mL) −0.006 ± 0.38 0.96 0.271 ± 1.413 0.53
LPS (EU/mL) −0.001 ± 0.006 >0.99 −0.041 ± 0.059 0.04 *
Gln suppl, L-glutamine supplementation. Ala suppl, alanine supplementation. BMI, body mass index. WC, waist
circumference. TNF-α, tumor necrosis alpha. IL-1β, interleukin-1 beta. IL6, interleukin 6. LPS, lipopolysaccharide.
Data were expressed as the difference between before and after 14 days of supplementation with either Ala or Gln.
Serum parameters were obtained from overnight fasted volunteers. Data were expressed as mean ± SD. p-value
indicates the difference between before and after glutamine or alanine supplementation obtained by paired Student’s
t test under normality and the Wilcoxon Mann–Whitney test otherwise. * p indicates a significant difference before
the same amino acid supplementation.
 Nutrients 2019, 11, 536 6 of 16

Table 2. Differences in metabolic characteristics of obese volunteers before and after supplementation
with glutamine or alanine.

Obese Volunteers
Ala Suppl Difference between Gln Supplemented Difference
p-Value p-Value
After and Before (n = 7) between After and Before (n = 13)
Height (m)
Weight (kg) 0.3 ± 1.02 0.46 −0.21 ± 0.76 0.34
BMI (kg/m2 ) 0.12 ± 0.39 0.44 −0.08 ± 0.30 0.37
Serum amino acid levels (µmol/L) 110.97 ± 15.53 0.0001 105.99 ± 14.99 0.0001
WC (cm) 0.0 ± 1.19 >0.99 −2.61 ± 2.21 0.002 *
Glucose (mmol/L) 0.25 ± 0.28 0.06 0.03 ± 0.41 0.84
Insulin (µU/mL) 0.34 ± 0.94 0.27 −1.15 ± 1.7 0.037 *
TNF-α (pg/mL) 0.11 ± 0.63 0.67 −2.65 ± 4.18 0.09
IL-1β (pg/mL) 0.06 ± 0.45 0.74 −0.24 ± 0.74 0.41
IL-6 (pg/mL) 0.07 ± 0.21 0.37 −0.27 ± 0.74 0.22
LPS (EU/mL) −0.001 ± 0.01 0.78 −0.08 ± 0.12 0.08
Gln suppl, L-glutamine supplementation. Ala suppl, alanine supplementation. BMI, body mass index. WC, waist
circumference. TNF-α, tumor necrosis alpha. IL-1β, interleukin-1 beta. IL6, interleukin 6. LPS, lipopolysaccharide.
Data were expressed as the difference between before and after 14 days of supplementation with either Ala or
Gln. Serum parameters were obtained from volunteers who fasted overnight. Data were expressed as mean ± SD.
The p-value indicates the difference between before and after glutamine or alanine supplementation obtained by
paired Student’s t test under normality and Wilcoxon Mann–Whitney test otherwise. * p indicates a significant
difference before the same amino acid supplementation.

Regarding the obese group, no differences were observed in body weight and BMI after
supplementation with Gln or Ala. In a similar fashion with overweight subjects, supplementation
with glutamine induced a significant reduction in waist circumference, whereas Ala supplementation
did not alter this parameter (Table 2). No change in glycemia was observed in obese individuals after
Gln or Ala supplementation (Table 2). However, a significant reduction in insulin levels was found
in obese subjects after Gln supplementation (Table 2). No changes were observed in serum levels of
TNF-α, IL-1β, and IL-6, but LPS showed a nonsignificant reduction after supplementation with Gln
in obese subjects. In addition, Ala levels were increased in Ala supplemented volunteers and Gln
levels were also increased in Gln supplemented volunteers (Obese volunteers supplemented with Ala
Nutrients
Before2018, 11, xµmol/L
502.89 FOR PEER
±REVIEW 7 of
124.56 n = 7 and after 613.87 µmol/L ± 119.43 n = 7. Obese volunteers 17
were
supplemented with Gln before 415.53 µmol/L ± 19.09 n = 13 and after 521.52 µmol/L ± 25.98 n = 13
and supplemental data). There was a clear positive correlation between reductions in LPS circulating
levels and reductions in waist circumference after glutamine supplementation in overweight and obese
subjects (r = 0.629), as shown in Figure 1.

Positive
Figure1.1.Positive
Figure correlation
correlation betweenthethereduction
between reductionofofwaist
waistcircumference
circumference(WC)
(WC)and andserum
serum
lipopolysaccharide
lipopolysaccharide (LPS)
(LPS) of of obese
obese and
and overweight
overweight humans.
humans. TheThe Pearson
Pearson Correlation
Correlation testtest
waswas
usedused
(r
= (r = 0.629).
0.629).

3.2.Effects
3.2. EffectsofofGlutamine
GlutamineSupplementation
SupplementationononAnimal
AnimalCharacteristics
Characteristics
Ratson
Rats onHFD
HFDpresented
presentedhigher
higherbody
bodyweight
weightthan
thananimals
animalsthat
thatreceived
receivedstandard
standardrodent
rodentchow
chow
(C+vehicle). However, rats on HFD and glutamine supplementation (H+Gln) gain
(C+vehicle). However, rats on HFD and glutamine supplementation (H+Gln) gain much less weightmuch less weight
than rats on HFD treated with alanine (H+Ala) (Figure 2A). As expected, epididymal, retroperitoneal,
and mesenteric fat mass was increased in the H+Ala group compared to C+vehicle, but there was a
clear decrease in these fat pads in H+Gln groups (Figures 2B to 2D).
Nutrients 2019, 11, 536 7 of 16

than rats on HFD treated with alanine (H+Ala) (Figure 2A). As expected, epididymal, retroperitoneal,
and mesenteric fat mass was increased in the H+Ala group compared to C+vehicle, but there was a
clear decrease in these fat pads in H+Gln groups (Figure 2B–D).
Although rats on HFD had higher (p < 0.05) food intake (g) than rats on C+vehicle (24.05 ± 0.79,
n = 8), there
Figure 1. were nocorrelation
Positive differences in food
between theintake between
reduction HFD
of waist supplemented
circumference (WC)with
and glutamine
serum
± 0.51, n = 7) or (LPS)
(22.73lipopolysaccharide alanine ± 0.69,
(22.65and
of obese n = 10).humans. The Pearson Correlation test was used (r
overweight
= 0.629). to energy metabolism on HFD rats, glutamine supplementation (32.3 ± 1.7 mL/kg/min,
Related
n = 4) recovered the reduced O2 consumption (mL/kg/min) observed in the H+Ala group (24.0 ± 1.2,
n3.2. Effects
= 5) sinceofthe
Glutamine
C+ vehicle (35.0 ± 1.0, non
Supplementation Animal
= 4) Characteristics
displayed similar O2 consumption as the H+Gln group.
The RER was reduced (p < 0.05) in the H+Ala group (0.72 ± 0.01, n = 5) compared to the C+ vehicle
Rats on HFD presented higher body weight than animals that received standard rodent chow
group (0.89 ± 0.01, n = 4), but the supplementation with glutamine reduced the RER even more
(C+vehicle). However, rats on HFD and glutamine supplementation (H+Gln) gain much less weight
significantly, which was significantly lower when compared to the H+Ala group (H+Gln group:
than rats on HFD treated with alanine (H+Ala) (Figure 2A). As expected, epididymal, retroperitoneal,
0.79 ± 0.01, n = 4 vs. H+Ala group: (0.72 ± 0.01, n = 5), which indicates that these animals were
and mesenteric fat mass was increased in the H+Ala group compared to C+vehicle, but there was a
oxidizing predominantly fatty acids.
clear decrease in these fat pads in H+Gln groups (Figures 2B to 2D).
Serum insulin, leptin, TNF-α, LPS and IL-6 levels were higher in the H+Ala group and, after
supplementation with glutamine, there was a clear reduction in all these parameters (Table 3). In contrast,
adiponectin levels were reduced in the H+Ala group and increased in the H+Gln group (Table 3).

Figure 2.
Figure 2. Body
Body Weight
Weight (BW)
(BW) during
during 8-weeks
8-weeks ofof amino
amino acids
acids or
or vehicle
vehicle (water)
(water) supplementation
supplementation via via
gavage
gavage started at 4-weeks
4-weeks (A), epididymal
epididymal fat mass (B), retroperitoneal fat mass (C), and mesenteric
mass (B), retroperitoneal fat mass (C), and mesenteric
fat mass (D). Rats
fat Rats that
thatreceived
receivedchow
chowdiet
dietand
andvehicle (C+Veh,
vehicle (C+Veh,n =n8), ratsrats
= 8), fedfed
with a high
with fat diet
a high and
fat diet
and supplemented with Alanina (H+Ala, n = 10) and rats fed with a high-fat diet and supplemented
with glutamine (H+Gln, n = 7). Values were displayed as mean ± SEM. Two-Way-ANOVA with a
Bonferroni post-test was used in (A). One-Way-ANOVA with a Bonferroni post-test that was used
in (B–D). * p < 0.05 vs. C+Veh and vs. H+Gln. # p < 0.05 vs. C+Veh.
Nutrients 2019, 11, 536 8 of 16

Table 3. Animal’s metabolic characteristics.

C+vehicle H+Ala H+Gln

Insulin (ng/mL) 0.24 ± 0.01 (n = 5) 1.14 ±0.21 * (n = 8) 0.49 ±0.08 # (n = 6)
Adiponectin (µg/mL) 8.16 ± 0.29 (n = 5) 4.86 ± 0.22 * (n = 6) 6.78 ± 0.4 # (n = 6)
TNF alpha (pg/mL) 95.94 ± 7.06 (n = 4) 499.60 ± 63.23 * (n = 5) 250.00 ± 34.86 (n = 5)
IL6 (pg/mL) 349.50 ± 5.80 (n = 4) 609.50 ± 46.10 * (n = 5) 347.50 ± 12.90 (n = 4)
LPS (EU/mL) 0.464 ± 0.05 (n = 5) 1.15 ± 0.16 * (n = 6) 0.74 ± 0.06 (n = 6)
Data were expressed means ± SEM. * p < 0.05 vs. C+vehicle group and H+Gln; # p < 0.05 vs. C+vehicle
group. One-Way-ANOVA with the Bonferroni post-test was used for statistical analysis. GIR, steady-state
glucose infusion rates were obtained from averaged rates of 90–120 min of 10% unlabeled glucose infusion. HGP,
hepatic glucose production. TNF alpha, tumor necrosis alpha. IL-1 beta, interleukin-1 beta. IL-6, interleukin 6;
LPS, lipopolysaccharide.

3.3. Effect of Glutamine Supplementation on Insulin-Induced Glucose Uptake in Muscle and Adipose Tissue
and on the Suppression of Hepatic Glucose Output
By using the glucose clamp with tracer infusion, we investigated whole body insulin sensitivity
associated with the effect of insulin on hepatic glucose production and glucose uptake in muscle
and adipose tissue. The insulin sensitivity, which was determined by the glucose infusion rate (GIR)
during the clamp, was diminished in the H+Ala group compared to the other groups (Figure 3).
The hepatic glucose production after insulin infusion was less suppressed in the H+Ala group and
supplementation with glutamine recovery including the inhibitory effect of insulin on this parameter
(Figure 3). Insulin-induced glucose uptake in muscle was reduced in the H+Ala group and normalized
in the H+Gln group (Figure 3). In adipose tissue, HFD (H+Ala group) induced an increase in glucose
uptake, which confirms previous data that HFD induced insulin resistance in muscle and liver but not
in adipose
Nutrients 2018, tissue
11, x FOR[25,26]. However, the supplementation with glutamine (H+Gln group)
PEER REVIEW 9 ofdramatically
17
reduced glucose incorporation in adipose tissue to levels even lower than the controls, which indicates
clear insulin resistance in this tissue (Figure 3).

Figure
Figure3.3.
Steady-state glucose
Steady-state infusion
glucose rates (GIR)
infusion ratesobtained from averaged
(GIR) obtained fromrates of 90 torates
averaged 120 minutes
of 90 to 120 min
of
of 10% unlabeled glucose infusion (A), insulin-induced suppression of hepaticproduction
10% unlabeled glucose infusion (A), insulin-induced suppression of hepatic glucose glucose production
(HGP)
(HGP)(B),
(B),insulin-stimulated
insulin-stimulatedmuscle glucose
muscle uptakeuptake
glucose (C), and insulin-stimulated
(C), adipose glucose
and insulin-stimulated adipose glucose
uptake (D). Rats that received the chow diet and vehicle (C+Veh, n = 4), rats fed with a high-fat diet
uptake (D). Rats that received the chow diet and vehicle (C+Veh, n = 4), rats fed with a high-fat
and supplemented with Alanina (H+Ala, n = 6) and rats fed with a high-fat diet supplemented with
diet and supplemented with Alanina (H+Ala, n = 6) and rats fed with a high-fat diet supplemented
glutamine (H+Gln, n = 4). Values were displayed as mean ± SEM. One-Way-ANOVA with the
with glutamine (H+Gln, n = 4). Values were displayed as mean ± SEM. One-Way-ANOVA with the
Bonferroni post-test was used in A to D. * p < 0.05 vs C+Veh and H+Ala; ** p < 0.05 vs same group
Bonferroni
without insulinpost-test was #used
stimulation; in A
p < 0.05 vsto D. * p < 0.05 vs. C+Veh and H+Ala; ** p < 0.05 vs. same group
C+Veh.
without insulin stimulation; # p < 0.05 vs. C+Veh.
3.4. Insulin-Induced Insulin Signaling in the Tissues of Rats on HFD Supplemented with Glutamine
The insulin-induced Akt phosphorylation was reduced in the liver and muscle of rats in the
H+Ala group, and glutamine supplementation recovery Akt phosphorylation in these tissues.
However, in adipose tissue, insulin-induced Akt phosphorylation, which was increased in the H+Ala
group, did not show an increase in the H+Gln group, which indicates that glutamine
Nutrients 2019, 11, 536 9 of 16

3.4. Insulin-Induced Insulin Signaling in the Tissues of Rats on HFD Supplemented with Glutamine
The insulin-induced Akt phosphorylation was reduced in the liver and muscle of rats in the H+Ala
Nutrientsand
group, 2018,glutamine
11, x FOR PEER REVIEW
supplementation 10 ofin
recovery Akt phosphorylation in these tissues. However, 17

adipose tissue, insulin-induced Akt phosphorylation, which was increased in the H+Ala group, did
not show an increase in the H+Gln group, which indicates that glutamine supplementation prevented
increased insulin signaling (Figure 4).

Figure 4.
Figure 4. Insulin
Insulin signaling
signalingin inthe
theliver,
liver,muscle,
muscle,and
andadipose
adipose tissue.
tissue. Akt
Akt phosphorylation
phosphorylation in in response
response to
to insulin or saline (control) in the liver (A) or muscle (B) or adipose tissue (C) of rats
insulin or saline (control) in the liver (A) or muscle (B) or adipose tissue (C) of rats that received thethat received
the chow
chow diet vehicle
diet and and vehicle (C+Veh),
(C+Veh), rats fedrats feda with
with a high-fat
high-fat diet anddiet and supplemented
supplemented with Alaninawith(H+Ala),
Alanina
(H+Ala),
and andwith
rats fed rats afed with adiet
high-fat high-fat diet and supplemented
and supplemented with glutamine
with glutamine (H+Gln).(H+Gln).
Beta actin Beta
wasactin
usedwas
as
used
the as the control
loading loadingin control in allBeta
all tissues. tissues. Beta
actin wasactin
usedwas used as control
as loading loadingincontrol in all tissues.
all tissues.

To
To investigate
investigate whether
whether thethe reduced
reduced LPS LPS circulating
circulating levels
levels might
might contribute
contribute to
to improvements
improvements in in
insulin signaling in the liver and muscle
insulin signaling in the liver and muscle of the H+Glnof the H+Gln group, we observed nuclear NF-κB activity
nuclear NF-κB activity in in
tissues
tissues of
ofanimals
animalssupplemented
supplementedwith withglutamine.
glutamine.It It
is is
well known
well knownthat LPS
that actsacts
LPS by activating Toll-like
by activating Toll-
receptor 4 (TLR4), which induces NF-κB activation. In this regard, the measurement
like receptor 4 (TLR4), which induces NF-κB activation. In this regard, the measurement of nuclear of nuclear NF-κB
activity in tissues
NF-κB activity is an indirect
in tissues evaluation
is an indirect of TLR4ofactivation
evaluation by LPS.by
TLR4 activation The results
LPS. showed
The results that, inthat,
showed the
H+Ala group,group,
in the H+Ala NF-κBNF-κB
activity was increased,
activity and the
was increased, andsupplementation withwith
the supplementation glutamine reduced
glutamine the
reduced
activity of this
the activity nuclear
of this factor
nuclear in allintissues
factor studied
all tissues (Figure
studied 5). 5).
(Figure
Multiple mechanisms may account for insulin resistance in obesity, and most of them are linked to
inflammatory signaling. Since glutamine reduced the NF-κB activity in adipose tissue, we believe that
glutamine-induced insulin resistance in adipose tissue is not related to increased inflammation. In this
regard, we next investigated another mechanism of insulin resistance, which is a posttranslational
modification of insulin receptor substrates by O-linked N-acetylglucosamine (O-GlcNAc). We then
performed immunoprecipitation of tissue extracts with antibodies anti-IRS-1 followed by blots with
antibodies anti O-GlcNAc. The results showed that IRS-1 was greater associated with O-GlcNAc only
in the adipose tissue of H + Gln group, but not in the liver or muscle. (Figure 6). In parallel, there
was a clear reduction in IRS-1 tissue protein levels in adipose tissue of the H+Ala group, which was
reversed after glutamine supplementation (Figure 6).
Nutrients 2018, 11, x FOR PEER REVIEW 11 of 17

Nutrients 2019, 11, 536 10 of 16

Nutrients 2018, 11, x FOR PEER REVIEW 12 of 17

Figure 5.
Figure 5. Nuclear
Nuclear factor
factor kappa
kappa BB (NF-κB)
(NF-κB) in
in the
the liver
liver (A),
(A), muscle
muscle (B),
(B), and
and adipose
adipose tissue
tissue (C).
(C). Rats
Rats that
that
received the chow diet and vehicle (C+Veh, n = 5), rats fed with a high-fat diet and supplemented
received the chow diet and vehicle (C+Veh, n = 5), rats fed with a high-fat diet and supplemented with with
Alanina (H+Ala;
Alanina (H+Ala; n == 5),
5), and
andrats
ratsfed
fedwith
withaahigh-fat
high-fatdiet
dietand
andsupplemented
supplementedwith withglutamine
glutamine(H+Gln,
(H+Gln, n
n= =5).5).Values
Valueswere
weredisplayed
displayedas asmean
mean ±±SEM.
SEM.One-Way-ANOVA
One-Way-ANOVA with with the Bonferroni post-test
post-test was
was
used. ** pp << 0.05 vs.
used. vs C+Veh
C+Vehand andH+Gln.
H+Gln.

Multiple mechanisms may account for insulin resistance in obesity, and most of them are linked
to inflammatory signaling. Since glutamine reduced the NF-κB activity in adipose tissue, we believe
that glutamine-induced insulin resistance in adipose tissue is not related to increased inflammation.
In this regard, we next investigated another mechanism of insulin resistance, which is a
posttranslational modification of insulin receptor substrates by O-linked N-acetylglucosamine (O-
GlcNAc). We then performed immunoprecipitation of tissue extracts with antibodies anti-IRS-1
followed by blots with antibodies anti O-GlcNAc. The results showed that IRS-1 was greater
associated with O-GlcNAc only in the adipose tissue of H + Gln group, but not in the liver or muscle.
(Figure 6). In parallel, there was a clear reduction in IRS-1 tissue protein levels in adipose tissue of
the H+Ala group, which was reversed after glutamine supplementation (Figure 6).

Figure 6. Insulin receptor substrate 1 (IRS1) association with N-O-linked glycosamine in the liver (A),
muscle (B), and adipose tissue (C). Rats that received a chow diet and vehicle (C+Veh; n = 2), rats
fed with high-fat diet and supplemented with Alanina (H+Ala; n = 2), and rats fed with a high-fat
diet and supplemented with glutamine (H+Gln; n = 2). Values were displayed as mean ± SEM.
One-Way-ANOVA with the Bonferroni post-test was used. * p < 0.05 vs. C+Veh and H+Ala.

4. Discussion
Here, we showed that glutamine supplementation to rats treated with HFD reduces weight gain
and improves insulin action and signaling in the liver and muscle. These beneficial effects seem to
be associated with tissue-specific insulin resistance in adipose tissue, which prevents the increase
in adipose mass in these mice. In a proof of concept study, we also showed that, in humans who
Nutrients 2019, 11, 536 11 of 16

are overweight or obese, oral Gln supplementation reduces waist circumference in parallel with a
reduction in circulating LPS, which suggests a modulation of microbiota and/or an intestinal barrier.
It is important to mention that we used alanine as a control to give the same amount of calories to
the group, which received glutamine. In addition, alanine was chosen because it is the second most
abundant circulating amino acid, is produced in muscle and metabolized in the liver as glutamine,
and transports ammonia from the muscle to the liver to produce urea [17]. Furthermore, alanine did
not significantly affect muscle metabolism or renal and hepatic function [35].
Although in humans, Gln supplementation reduced waist circumference, it did not change body
weight and BMI of overweight and obese subjects. The absence of changes on body weight and BMI
in humans may be due to the short duration of supplementation. However, waist circumference
measurements are a viable and reliable way to measure abdominal fat mass and represent mostly
visceral adipose tissue [36,37]. Visceral adipose tissue loss is associated with improved insulin
resistance, which decreases the risk of type 2 diabetes development [37–40].
Both overweight humans and animals on high fat diet with glutamine supplementation reduced
circulating lipopolysaccharide levels. The gastrointestinal tract is the main source of LPS, because
of its massive bacterial load compared to other anatomical sites [41] and there is a direct and
strong association between plasma LPS levels and the degree of intestinal permeability, in different
pathological conditions [42]. This translocation of LPS into the plasma leads to binding of LPS to TLR4,
which will induce the activation of the NF-κB pathway in different tissues, which results in systemic
inflammation [43–46] and insulin resistance [42]. In mice, the subcutaneous infusion of LPS induces
glucose intolerance, insulin resistance, and a very interesting increase in body weight accompanied
by increases in adipose tissue [11,47]. The mechanism by which glutamine reduces circulating LPS
levels is not completely understood but might involve modulation of intestinal microbiota and or
changes in intestinal permeability. Recently, our group demonstrated that L-glutamine altered gut
microbiota by decreasing the Firmicutes/Bacteroidetes ratio [21], which is considered a biomarker for
obesity. Since we used the same protocol to supplement L-glutamine in the present study, we can,
thus, suggest that glutamine may have modulated the microbiota. Taken together, these data with our
results suggests that glutamine supplementation may induce changes in intestinal microbiota and in
intestinal permeability, which may account for reduced circulating levels of LPS in humans and the
animal model, which contributes to improved insulin action.
Previous data demonstrated that in the first months of HFD administration occurred insulin
resistance in the liver and muscle, but not in the adipose tissue of rats and mice [25,26].
This tissue-specific protection from insulin resistance in adipose tissue may have an important role
in the development of obesity because it allows insulin to increase glucose uptake and synthesize
triglycerides in this tissue [7]. However, the inflammatory process that develops in adipose tissue with
infiltration of macrophages and the consequent increase in circulating cytokines certainly contribute
to worsening the systemic insulin resistance [48–50]. In this regard, previous data showed that
tissue-specific knockout of the insulin receptor in adipose tissue (FIRKO mice) protects the animal
from diet-induced insulin resistance [24]. Based on previous data that glutamine supplementation may
induce insulin resistance in adipose cell lines but not in muscle cell lines [27–29], we used glutamine
supplementation to induce insulin resistance in the adipose tissue of an HFD animal. Our data showed
that we could develop a model of adipose tissue-specific insulin resistance in rats by protecting these
animals against diet-induced systemic insulin resistance, which is similar to what was described for
the FIRKO mice.
We also investigated the molecular mechanism by which glutamine induces insulin resistance
only in adipose tissue and reduces glucose uptake and triglycerides synthesis in this tissue.
Several mechanisms have been described to explain insulin resistance, and low-grade inflammation
represents one of the most important of these mechanisms in obesity [48,49]. However, the adipose
insulin resistance induced by glutamine was not related to inflammation, because we found a reduced
circulating LPS in humans and the animal model. Furthermore, in adipose tissue of rats, there was a
Nutrients 2019, 11, 536 12 of 16

decrease in NF-κB activity, which indicates a clear reduction in an inflammatory milieu. The insulin
resistance in adipose tissue of animals on a high fat diet supplemented with glutamine was associated
with a reduction of ~50% of fat mass, which suggests that these mechanisms may be linked. One
possibility is that GLN increases the activity of the hexosamine pathway (HBP) [51]. GLN is an
intermediary substrate to generate glucosamine-6-phosphate via HBP, which is further metabolized
to UDP-GlcNAc (UDP-N-acetylglucosamine). UDP-GlcNAc causes posttranslational modification
of proteins, which leads to lower lipogenesis and reduced adipose mass [51]. It is well known that
the hexosamine pathway can induce insulin resistance through direct post-translational modification
of key insulin signaling proteins, via O-linked glycosylation on serine and threonine residues with
the GlcNAc moiety [51]. Our results demonstrating increased IRS1/O-GlcNAc association in adipose
tissue suggest that O-linked GlcNAc post-translational modification may induce insulin resistance in
this tissue.
Another important point that should be considered in animals supplemented with glutamine is
the increase in adiponectin levels associated with an increase in energy expenditure and a decrease in
RER, which indicates that these animals were largely using fatty acids as an energy source. In addition,
to act as a glucose-lowering adipokine [52–57], adiponectin also increases fatty acids oxidation [58].
Our data showing an increase in circulating adiponectin levels after glutamine supplementation may
explain the increased energy expenditure and fat oxidation, which reinforces the protection from
diet-induced obesity in these animals.
Our study has some limitations such as the short time of glutamine supplementation in humans
and the absence of a gold standard method to measure insulin sensitivity, and of an oral glucose
tolerance test in humans. In addition, 24-hour dietary recall and our assessment of the physical activity
level have limitations.
Our data show that glutamine supplementation, in animals and humans, was accompanied by
an increase in plasma glutamine levels and in rat tissue by an increase in IRS1/O-GlcNAc in adipose
tissue but not in the liver and the muscle tissue. This action of glutamine mainly in adipose tissue may
be a consequence of differential mechanisms involved in the glutamine transport among tissues [59],
but, certainly, this point deserves a deep investigation.

5. Conclusions
In summary, our data showed that glutamine supplementation could reduce the insulin action
and glucose uptake in fat and adipose mass, which improves the insulin action and signaling in the
liver and muscle of rats. In addition, a proof of concept study showed that, in humans, glutamine
supplementation for a short time is accompanied by a reduction in waist circumference, in circulating
LPS. Our preliminary data suggest that further investigations with glutamine supplementation should
be performed for longer periods. In addition, it would be interesting to investigate whether glutamine
is also able to induce insulin resistance in the adipose tissue of humans, which may have some
long-term detrimental consequences.

Supplementary Materials: The following are available online at http://www.mdpi.com/2072-6643/11/3/536/s1,

Table S1: Metabolic characteristics of overweight volunteers before and after supplementation with glutamine or
alanine. Table S2: Metabolic characteristics of obese volunteers before and after supplementation with glutamine
or alanine.
Author Contributions: P.O.P. and M.J.A.S. conceived the study. G.Z.R.; P.O.P. and M.J.A.S. designed experiments.
K.Y.A.; S.K.R.; A.Z.Z.d.S.; F.T.; M.E.M.; O.P.Z.; H.B.A.; D.G.; G.Z.R. performed all human and animal experiments,
and analyzed data. P.O.P. and M.J.A.S. wrote the manuscript. All authors read and edited the manuscript.
Funding: CNPq (National Council for Scientific and Technological Development) 465693/2014-8; FAPESP
(Sao Paulo Research Foundation) 2017/18498-6.
Conflicts of Interest: The authors declare no conflict of interest.
Nutrients 2019, 11, 536 13 of 16

References
1. Fruh, S.M. Obesity: Risk factors, complications, and strategies for sustainable long-term weight management.
J. Am. Assoc. Nurse Pract. 2017, 29, S3–S14. [CrossRef] [PubMed]
2. Malik, V.S.; Willett, W.C.; Hu, F.B. Global obesity: Trends, risk factors and policy implications.
Nat. Rev. Endocrinol. 2013, 9, 13–27. [CrossRef] [PubMed]
3. Alberti, K.G.; Eckel, R.H.; Grundy, S.M.; Zimmet, P.Z.; Cleeman, J.I.; Donato, K.A.; Fruchart, J.C.; James, W.P.;
Loria, C.M.; Smith, S.C., Jr.; et al. Harmonizing the metabolic syndrome: A joint interim statement of the
International Diabetes Federation Task Force on Epidemiology and Prevention; National Heart, Lung, and
Blood Institute; American Heart Association; World Heart Federation; International Atherosclerosis Society;
and International Association for the Study of Obesity. Circulation 2009, 120, 1640–1645. [PubMed]
4. Collaboration, N.C. Trends in adult body-mass index in 200 countries from 1975 to 2014: A pooled analysis
of 1698 population-based measurement studies with 19.2 million participants. Lancet 2016, 387, 1377–1396.
5. Sellayah, D.; Cagampang, F.R.; Cox, R.D. On the evolutionary origins of obesity: A new hypothesis.
Endocrinology 2014, 155, 1573–1588. [CrossRef] [PubMed]
6. Burhans, M.S.; Hagman, D.K.; Kuzma, J.N.; Schmidt, K.A.; Kratz, M. Contribution of Adipose Tissue
Inflammation to the Development of Type 2 Diabetes Mellitus. Compr. Physiol. 2018, 9, 1–58. [PubMed]
7. Czech, M.P. Insulin action and resistance in obesity and type 2 diabetes. Nat. Med. 2017, 23, 804–814.
[CrossRef] [PubMed]
8. Prada, P.O.; Saad, M.J. Tyrosine kinase inhibitors as novel drugs for the treatment of diabetes. Expert Opin.
Investig. Drugs 2013, 22, 751–763. [CrossRef] [PubMed]
9. Czech, M.P. Macrophages dispose of catecholamines in adipose tissue. Nat. Med. 2017, 23, 1255–1257.
[CrossRef] [PubMed]
10. Bagarolli, R.A.; Tobar, N.; Oliveira, A.G.; Araújo, T.G.; Carvalho, B.M.; Rocha, G.Z.; Vecina, J.F.; Calisto, K.;
Guadagnini, D.; Prada, P.O.; et al. Probiotics modulate gut microbiota and improve insulin sensitivity in
DIO mice. J. Nutr. Biochem. 2017, 50, 16–25. [CrossRef] [PubMed]
11. Cani, P.D.; Amar, J.; Iglesias, M.A.; Poggi, M.; Knauf, C.; Bastelica, D.; Neyrinck, A.M.; Fava, F.; Tuohy, K.M.;
Chabo, C.; et al. Metabolic endotoxemia initiates obesity and insulin resistance. Diabetes 2007, 56, 1761–1772.
[CrossRef] [PubMed]
12. Carvalho, B.M.; Saad, M.J. Influence of gut microbiota on subclinical inflammation and insulin resistance.
Mediators Inflamm. 2013, 2013, 986734. [CrossRef] [PubMed]
13. Forsythe, L.K.; Wallace, J.M.; Livingstone, M.B. Obesity and inflammation: The effects of weight loss.
Nutr. Res. Rev. 2008, 21, 117–133. [CrossRef] [PubMed]
14. Friedman, J.M. Obesity in the new millennium. Nature 2000, 404, 632–634. [CrossRef] [PubMed]
15. Andreasen, A.S.; Pedersen-Skovsgaard, T.; Mortensen, O.H.; van Hall, G.; Moseley, P.L.; Pedersen, B.K.
The effect of glutamine infusion on the inflammatory response and HSP70 during human experimental
endotoxaemia. Crit. Care 2009, 13, R7. [CrossRef] [PubMed]
16. Beutheu, S.; Ouelaa, W.; Guérin, C.; Belmonte, L.; Aziz, M.; Tennoune, N.; Bôle-Feysot, C.; Galas, L.;
Déchelotte, P.; Coëffier, M. Glutamine supplementation, but not combined glutamine and arginine
supplementation, improves gut barrier function during chemotherapy-induced intestinal mucositis in
rats. Clin. Nutr. 2014, 33, 694–701. [CrossRef] [PubMed]
17. Curi, R.; Lagranha, C.J.; Doi, S.Q.; Sellitti, D.F.; Procopio, J.; Pithon-Curi, T.C.; Corless, M.; Newsholme, P.
Molecular mechanisms of glutamine action. J. Cell. Physiol. 2005, 204, 392–401. [CrossRef] [PubMed]
18. Lin, Z.; Cai, F.; Lin, N.; Ye, J.; Zheng, Q.; Ding, G. Effects of glutamine on oxidative stress and nuclear
factor-kappaB expression in the livers of rats with nonalcoholic fatty liver disease. Exp. Ther. Med. 2014, 7,
365–370. [CrossRef] [PubMed]
19. Shanware, N.P.; Mullen, A.R.; De Berardinis, R.J.; Abraham, R.T. Glutamine: Pleiotropic roles in tumor
growth and stress resistance. J. Mol. Med. 2011, 89, 229–236. [CrossRef] [PubMed]
20. Zhou, X.; Wu, X.; Yin, Y.; Zhang, C.; He, L. Preventive oral supplementation with glutamine and arginine
has beneficial effects on the intestinal mucosa and inflammatory cytokines in endotoxemic rats. Amino Acids
2012, 43, 813–821. [CrossRef] [PubMed]
Nutrients 2019, 11, 536 14 of 16

21. De Souza, A.Z.; Zambom, A.Z.; Abboud, K.Y.; Reis, S.K.; Tannihao, F.; Guadagnini, D.; Saad, M.J.; Prada, P.O.
Oral supplementation with L-glutamine alters gut microbiota of obese and overweight adults: A pilot study.
Nutrition 2015, 31, 884–889. [CrossRef] [PubMed]
22. Laviano, A.; Molfino, A.; Lacaria, M.T.; Canelli, A.; De Leo, S.; Preziosa, I.; Rossi Fanelli, F. Glutamine
supplementation favors weight loss in nondieting obese female patients. A pilot study. Eur. J. Clin. Nutr.
2014, 68, 1264–1266. [CrossRef] [PubMed]
23. Mansour, A.; Mohajeri-Tehrani, M.R.; Qorbani, M.; Heshmat, R.; Larijani, B.; Hosseini, S. Effect of glutamine
supplementation on cardiovascular risk factors in patients with type 2 diabetes. Nutrition 2015, 31, 119–126.
[CrossRef] [PubMed]
24. Bluher, M.; Michael, M.D.; Peroni, O.D.; Ueki, K.; Carter, N.; Kahn, B.B.; Kahn, C.R. Adipose tissue selective
insulin receptor knockout protects against obesity and obesity-related glucose intolerance. Dev. Cell 2002, 3,
25–38. [CrossRef]
25. Prada, P.O.; Zecchin, H.G.; Gasparetti, A.L.; Torsoni, M.A.; Ueno, M.; Hirata, A.E.; Corezola do Amaral, M.E.;
Hoer, N.F.; Boschero, A.C.; Saad, M.J. Western diet modulates insulin signaling, c-Jun N-terminal kinase
activity, and insulin receptor substrate-1ser307 phosphorylation in a tissue-specific fashion. Endocrinology
2005, 146, 1576–1587. [CrossRef] [PubMed]
26. Thirone, A.C.; Carvalheira, J.B.; Hirata, A.E.; Velloso, L.A.; Saad, M.J. Regulation of Cbl-associated
protein/Cbl pathway in muscle and adipose tissues of two animal models of insulin resistance. Endocrinology
2004, 145, 281–293. [CrossRef] [PubMed]
27. Marshall, S.; Garvey, W.T.; Traxinger, R.R. New insights into the metabolic regulation of insulin action and
insulin resistance: Role of glucose and amino acids. FASEB J. 1991, 5, 3031–3036. [CrossRef] [PubMed]
28. Traxinger, R.R.; Marshall, S. Role of amino acids in modulating glucose-induced desensitization of the
glucose transport system. J. Biol. Chem. 1989, 264, 20910–20916. [PubMed]
29. Tremblay, F.; Marette, A. Amino acid and insulin signaling via the mTOR/p70 S6 kinase pathway. A negative
feedback mechanism leading to insulin resistance in skeletal muscle cells. J. Biol. Chem. 2001, 276,
38052–38060. [PubMed]
30. Garcia, R.F.; Gazola, V.A.; Barrena, H.C.; Hartmann, E.M.; Berti, J.; Toyama, M.H.; Boschero, A.C.;
Carneiro, E.M.; Manso, F.C.; Bazotte, R.B. Blood amino acids concentration during insulin induced
hypoglycemia in rats: The role of alanine and glutamine in glucose recovery. Amino Acids 2007, 33, 151–155.
[CrossRef] [PubMed]
31. Reagan-Shaw, S.; Nihal, M.; Ahmad, N. Dose translation from animal to human studies revisited. FASEB J.
2008, 22, 659–661. [CrossRef] [PubMed]
32. Hirabara, S.M.; Silveira, L.R.; Alberici, L.C.; Leandro, C.V.; Lambertucci, R.H.; Polimeno, G.C.; Cury
Boaventura, M.F.; Procopio, J.; Vercesi, A.E.; Curi, R. Acute effect of fatty acids on metabolism and
mitochondrial coupling in skeletal muscle. Biochim. Biophys. Acta 2006, 1757, 57–66. [CrossRef] [PubMed]
33. Saad, M.J.; Folli, F.; Araki, E.; Hashimoto, N.; Csermely, P.; Kahn, C.R. Regulation of insulin receptor, insulin
receptor substrate-1 and phosphatidylinositol 3-kinase in 3T3-F442A adipocytes. Effects of differentiation,
insulin, and dexamethasone. Mol. Endocrinol. 1994, 8, 545–557. [PubMed]
34. Thirone, A.C.; Carvalho, C.R.; Saad, M.J. Growth hormone stimulates the tyrosine kinase activity of JAK2
and induces tyrosine phosphorylation of insulin receptor substrates and Shc in rat tissues. Endocrinology
1999, 140, 55–62. [CrossRef] [PubMed]
35. Saunders, B.; Franchi, M.; de Oliveira, L.F.; da Eira Silva, V.; da Silva, R.P.; de Salles Painelli, V.; Costa, L.A.R.;
Sale, C.; Harris, R.C.; Roschel, H.; et al. 24-Week beta-alanine ingestion does not affect muscle taurine or
clinical blood parameters in healthy males. Eur. J. Nutr. 2018. [CrossRef] [PubMed]
36. Bonora, E. Relationship between regional fat distribution and insulin resistance. Int. J. Obes. Relat.
Metab. Disord. 2000, 24 (Suppl. 2), S32–S35. [CrossRef] [PubMed]
37. Ness-Abramof, R.; Apovian, C.M. Waist circumference measurement in clinical practice. Nutr. Clin. Pract.
2008, 23, 397–404. [CrossRef] [PubMed]
38. Nilsson, G.; Hedberg, P.; Jonason, T.; Lonnberg, I.; Tenerz, A.; Forberg, R.; Ohrvik, J. Waist circumference
alone predicts insulin resistance as good as the metabolic syndrome in elderly women. Eur. J. Intern. Med.
2008, 19, 520–526. [CrossRef] [PubMed]
Nutrients 2019, 11, 536 15 of 16

39. Staiano, A.E.; Reeder, B.A.; Elliott, S.; Joffres, M.R.; Pahwa, P.; Kirkland, S.A.; Paradis, G.; Katzmarzyk, P.T.
Body mass index versus waist circumference as predictors of mortality in Canadian adults. Int. J. Obes. 2012,
36, 1450–1454. [CrossRef] [PubMed]
40. Vogeser, M.; Konig, D.; Frey, I.; Predel, H.G.; Parhofer, K.G.; Berg, A. Fasting serum insulin and the
homeostasis model of insulin resistance (HOMA-IR) in the monitoring of lifestyle interventions in obese
persons. Clin. Biochem. 2007, 40, 964–968. [CrossRef] [PubMed]
41. Kelly, C.J.; Colgan, S.P.; Frank, D.N. Of microbes and meals: The health consequences of dietary endotoxemia.
Nutr. Clin. Pract. 2012, 27, 215–225. [CrossRef] [PubMed]
42. Manco, M.; Putignani, L.; Bottazzo, G.F. Gut microbiota, lipopolysaccharides, and innate immunity in the
pathogenesis of obesity and cardiovascular risk. Endocr. Rev. 2010, 31, 817–844. [CrossRef] [PubMed]
43. Brenchley, J.M.; Price, D.A.; Schacker, T.W.; Asher, T.E.; Silvestri, G.; Rao, S.; Kazzaz, Z.; Bornstein, E.;
Lambotte, O.; Altmann, D.; et al. Microbial translocation is a cause of systemic immune activation in chronic
HIV infection. Nat. Med. 2006, 12, 1365–1371. [CrossRef] [PubMed]
44. Bukh, A.R.; Melchjorsen, J.; Offersen, R.; Jensen, J.M.; Toft, L.; Stovring, H.; Ostergaard, L.; Tolstrup, M.;
Sogaard, O.S. Endotoxemia is associated with altered innate and adaptive immune responses in untreated
HIV-1 infected individuals. PLoS ONE 2011, 6, e21275. [CrossRef] [PubMed]
45. Schaller, S.J.; Anstey, M.; Blobner, M.; Edrich, T.; Grabitz, S.D.; Gradwohl-Matis, I.; Heim, M.; Houle, T.;
Kurth, T.; Latronico, N.; et al. Early, goal-directed mobilisation in the surgical intensive care unit:
A randomised controlled trial. Lancet 2016, 388, 1377–1388. [CrossRef]
46. Sun, L.; Yu, Z.; Ye, X.; Zou, S.; Li, H.; Yu, D.; Wu, H.; Chen, Y.; Dore, J.; Clement, K.; et al. A marker of
endotoxemia is associated with obesity and related metabolic disorders in apparently healthy Chinese.
Diabetes Care 2010, 33, 1925–1932. [CrossRef] [PubMed]
47. Creely, S.J.; McTernan, P.G.; Kusminski, C.M.; Fisher, F.M.; Da Silva, N.F.; Khanolkar, M.; Evans, M.;
Harte, A.L.; Kumar, S. Lipopolysaccharide activates an innate immune system response in human adipose
tissue in obesity and type 2 diabetes. Am. J. Physiol. Endocrinol. Metab. 2007, 292, E740–E747. [CrossRef]
[PubMed]
48. Hotamisligil, G.S. Inflammatory pathways and insulin action. Int. J. Obes. Relat. Metab. Disord. 2003, 27
(Suppl. 3), S53–S55. [CrossRef] [PubMed]
49. Hotamisligil, G.S. Inflammation and metabolic disorders. Nature 2006, 444, 860–867. [CrossRef] [PubMed]
50. Wellen, K.E.; Hotamisligil, G.S. Obesity-induced inflammatory changes in adipose tissue. J. Clin. Investig.
2003, 112, 1785–1788. [CrossRef] [PubMed]
51. Patti, M.E.; Virkamaki, A.; Landaker, E.J.; Kahn, C.R.; Yki-Jarvinen, H. Activation of the hexosamine pathway
by glucosamine in vivo induces insulin resistance of early postreceptor insulin signaling events in skeletal
muscle. Diabetes 1999, 48, 1562–1571. [CrossRef] [PubMed]
52. Berg, A.H.; Combs, T.P.; Du, X.; Brownlee, M.; Scherer, P.E. The adipocyte-secreted protein Acrp30 enhances
hepatic insulin action. Nat. Med. 2001, 7, 947–953. [CrossRef] [PubMed]
53. Berg, A.H.; Combs, T.P.; Scherer, P.E. ACRP30/adiponectin: An adipokine regulating glucose and lipid
metabolism. Trends Endocrinol. Metab. 2002, 13, 84–89. [CrossRef]
54. Kubota, N.; Terauchi, Y.; Yamauchi, T.; Kubota, T.; Moroi, M.; Matsui, J.; Eto, K.; Yamashita, T.; Kamon, J.;
Satoh, H.; et al. Disruption of adiponectin causes insulin resistance and neointimal formation. J. Biol. Chem.
2002, 277, 25863–25866. [CrossRef] [PubMed]
55. Maeda, N.; Shimomura, I.; Kishida, K.; Nishizawa, H.; Matsuda, M.; Nagaretani, H.; Furuyama, N.;
Kondo, H.; Takahashi, M.; Arita, Y.; et al. Diet-induced insulin resistance in mice lacking
adiponectin/ACRP30. Nat. Med. 2002, 8, 731–737. [CrossRef] [PubMed]
56. Yamauchi, T.; Yamauchi, T.; Kamon, J.; Ito, Y.; Tsuchida, A.; Yokomizo, T.; Kita, S.; Sugiyama, T.; Miyagishi, M.;
Hara, K.; Tsunoda, M.; et al. Cloning of adiponectin receptors that mediate antidiabetic metabolic effects.
Nature 2003, 423, 762–769. [CrossRef] [PubMed]
57. Yatagai, T.; Nagasaka, S.; Taniguchi, A.; Fukushima, M.; Nakamura, T.; Kuroe, A.; Nakai, Y.; Ishibashi, S.
Hypoadiponectinemia is associated with visceral fat accumulation and insulin resistance in Japanese men
with type 2 diabetes mellitus. Metabolism 2003, 52, 1274–1278. [CrossRef]
Nutrients 2019, 11, 536 16 of 16

58. Yamauchi, T.; Kamon, J.; Minokoshi, Y.; Ito, Y.; Waki, H.; Uchida, S.; Yamashita, S.; Noda, M.; Kita, S.; Ueki, K.;
et al. Adiponectin stimulates glucose utilization and fatty-acid oxidation by activating AMP-activated
protein kinase. Nat. Med. 2002, 8, 1288–1295. [CrossRef] [PubMed]
59. Ritchie, J.W.; Baird, F.E.; Christie, G.R.; Stewart, A.; Low, S.Y.; Hundal, H.S.; Taylor, P.M. Mechanisms of
glutamine transport in rat adipocytes and acute regulation by cell swelling. Cell. Physiol. Biochem. 2001, 11,
259–270. [CrossRef] [PubMed]

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).