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ORIGINAL ARTICLE
Inflammation and insulin resistance exert dual effects on adipose
tissue tumor protein 53 expression
FJ Ortega
1,2
, JM Moreno-Navarrete
1,2
, D Mayas
1,3
, M Serino
4
, JI Rodriguez-Hermosa
5
, W Ricart
1,2
, E Luche
4
, R Burcelin
4
, FJ Tinahones
1,3
,
GFru¨ hbeck
1,6
, G Mingrone
7
and JM Ferna´ndez-Real
1,2
OBJECTIVE: The purpose of this study was to investigate the expression of human adipose tissue protein 53 (p53) in subjects who
varied widely in terms of obesity and insulin resistance. We also analyzed different in vivo and in vitro models to try to comprehend
the associations found in humans.
METHODS: p53 was analyzed in human adipose and isolated adipocytes, in high fat-fed and GLP-1R KO mice, during in vitro
adipogenesis, and in adipocytes after high glucose, rosiglitazone and inflammatory conditions. The effects of surgery-induced
weight loss and ex vivo metformin were also evaluated.
RESULTS: Omental (OM) p53 gene expression ( þ 27%, P ¼ 0.001) and protein ( þ 11%, P ¼ 0.04) were increased in obese subjects
and high fat diet-induced obese mice ( þ 86%, P ¼ 0.018). Although the obesity-associated inflammatory milieu was associated with
increased OM p53, this was negatively related to insulin resistance and glycated hemoglobin, and positively with biomarkers for
insulin sensitivity. Multiple linear regression analyses revealed that glycated hemoglobin (Po0.0001) and body mass index
(P ¼ 0.048) contributed independently to explain 13.7% (Po0.0001) of the OM p53 variance. Accordingly, the improvement of
insulin sensitivity with surgery-induced weight loss ( þ 51%, P ¼ 0.01) and metformin ( þ 42%, P ¼ 0.02) led to increased adipose
p53. While the glucose-intolerant GLP-1R KO mice showed decreased mesenteric p53 ( 45.4%, P ¼ 0.017), high glucose led to
decreased p53 in pre-adipocytes ( 27%, Po0.0001). Inflammatory treatments led to increased p53 ( þ 35%, Po0.0001), while
Rs downregulated this expression ( 40%, P ¼ 0.005) in mature adipocytes.
CONCLUSION: Inflammation and insulin resistance exert dual effects on adipose p53, which seems to be the final result of these
opposing forces.
International Journal of Obesity advance online publication, 1 October 2013; doi:10.1038/ijo.2013.163
Keywords: tumor protein 53; adipocytes; adipose tissue; inflammation; insulin resistance; type 2 diabetes
INTRODUCTION
The tumor suppressor activity of the protein 53 (p53) has been
clearly established in the last three decades (see Levine and Oren
1
for a review). Less attention has been given to its possible
involvement in other biological processes such as cell metabolism
and development. Notably, inflammatory stress triggers the p53
response,
2
and p53 itself is also emerging as an important
regulator of metabolic homeostasis.
3
Both adipose tissue and systemic inflammation are up-
regulated in obese subjects, having a crucial role in the
progression of metabolic disturbances.
4,5
Omental (OM) adipose
tissue dysfunction is known to act as a source of inflammatory-
related factors in individuals with central obesity.
6
On the other
hand, adipose tissue senescence has been recently recognized to
be linked to inflammation in obese subjects with shortened
telomeres.
5,7
Minamino et al.
8
recently reported that p53 in mice adipose
tissue was closely associated with cellular aging, a new target for
the treatment of diabetes. These authors used Ay mice, known to
develop obesity and diabetes in the context of increased oxidative
stress and pro-inflammatory cytokines.
8
They demonstrated the
induction of senescence-like changes (for example, increased p53
and shortened telomeres) in adipose tissue from these mice.
8
Results from this study suggested that p53 in adipose tissue may
lead to increased oxidative stress-induced nuclear transcription
factor (NF)-kB activation and the induction of inflammatory
cytokines, promoting at the end the development of insulin
resistance in mice.
Recent findings also demonstrate that p53 modulates the
coordinated interplay between proliferation and differentiation in
many cell lineages,
9–11
including adipocytes,
12
having unsuspected
effects on cellular metabolism.
13,14
Molchadsky et al.
15
demonstrated
that p53 has opposing roles in a cell fate-dependent manner,
inhibiting adipogenesis but promoting muscle differentiation. In vitro
and in vivo studies have also provided interesting insights into the
association of p53 with adipocytes’ fate decisions (see Bazuine et al.
12
for a review), its involvement in adipose tissue dysfunction
8
and in
metabolic homeostasis.
1
CIBER de la Fisiopatologı
´
a de la Obesidad y Nutricio
´
n (CIBERobn, CB06/03/0010), and Instituto de Salud Carlos III (ISCIII), Santiago de Compostela, Spain;
2
Service of Diabetes,
Endocrinology and Nutrition (UDEN), Institut d’Investigacio
´
Biome
`
dica de Girona (IdIBGi), Hospital ‘‘Dr Josep Trueta’’ of Girona, Girona, Spain;
3
Service of Endocrinology and
Nutrition, Hospital Clı
´
nico Universitario Virgen de Victoria de Ma
´
laga, Ma
´
laga, Spain;
4
INSERM Unite
´
858, Institut de Me
´
decine Mole
´
culaire de Rangueil, Universite
´
Paul Sabatier,
IFR31, Toulouse, France;
5
Department of Surgery, Institut d’Investigacio
´
Biome
`
dica de Girona (IdIBGi), Girona, Spain;
6
Department of Endocrinology & Nutrition, Clı
´
nica
Universidad de Navarra–CIBERobn CBO6/03/1014, Pamplona, Spain and
7
Institute of Internal Medicine, Catholic University of Rome, Rome, Italy. Correspondence:
Dr JM Ferna
´
ndez-Real or Dr FJ Ortega, Service of Diabetes, Endocrinology and Nutrition (UDEN), Institut d’Investigacio
´
Biome
`
dica de Girona (IdIBGi), Hospital ‘‘Dr Josep Trueta’’ of
Girona, Carretera de Franc¸a s/n, Girona 17007, Spain.
E-mail: jmfreal@idibgi.org or fortega@idibgi.org
Received 21 March 2013; revised 2 July 2013; accepted 5 August 2013; accepted article preview online 3 September 2013
International Journal of Obesity (2013), 1–9
&
2013 Macmillan Publishers Limited All rights reserved 0307-0565/13
www.nature.com/ijo
In fact, p53 is able to contribute to the regulation of many
cellular pathways, including glycolysis,
16
insulin sensitivity,
14
fatty
acid oxidation and mTOR signaling.
17
Overall, the ability of p53 to
respond to nutrient deficiencies in insulin-sensitive tissues is
consistent with its established function as a mediator of stress,
3
but p53 seems to have many pleiotropic effects,
16,18
which remain
to be elucidated in this context.
The challenge is to define the specific role of adipose tissue p53
in association with inflammation and the development of obesity
and obesity-related complications, such as insulin resistance and
type 2 diabetes. Very few data are available regarding the
expression of p53 and its modulating partner, murine double
minute 2 (MDM2, a proto-oncogene that is closely associated with
p53 gene products), in human adipose tissue. Thus, the purpose of
this study was to investigate the expression of p53 and MDM2 in
subcutaneous (SC) and OM adipose tissue from a large cohort of
subjects who varied widely in terms of obesity and insulin
resistance. We also analyzed different in vivo and in vitro models to
try to comprehend the associations found in human adipose
tissue.
MATERIALS AND METHODS
Human studies
Subjects and samples. A total of 193 OM and 113 SC adipose tissue
samples (76 paired fat samples) were obtained from human fat depots
during elective surgical procedures. These fat samples were provided from
a group of 230 subjects (76 men and 154 women) with a body mass index
between 18 and 70 kg m
2
who were invited to participate at the
Endocrinology Service of the Hospital Universitari de Girona Dr Josep
Trueta (Girona, Spain) and the Hospital Carlos Haya de Ma
´
laga (Ma
´
laga,
Spain). All subjects were of Caucasian origin and reported that their body
weight had been stable for at least 3 months before the study. They had
no systemic disease other than type 2 diabetes and/or obesity, and all were
free of any infections within the previous months before the study. All
subjects gave written informed consent after the purpose of the study was
explained to them. Approximately 5 g of SC and OM adipose tissue fresh
samples from 12 subjects (24 paired samples) was used to isolate stromal-
vascular cells and MAs, as previously described.
19
Anthropometric and analytical measurements. BMI was calculated as
weight (in kg) divided by height (in m) squared. Bioelectric impedance
and/or air-displacement plethysmoghraphy were used to estimate the
body fat composition in those subjects. According to these anthropometric
parameters, subjects were classified as non-obese (BMIo30.0 kg m
2
) and
obese (BMIX30.0 kg m
2
, and body fat % X25% for men and X35% for
women) subjects. Data from a standard 75-g oral glucose tolerance test,
performed after an overnight fast, and venous blood samples that were
drawn at time points zero, 30, 60, 90 and 120 min for the determination of
plasma glucose concentrations, were available for a random subpopulation
of 28 participants (8 men and 20 women). Area under the curve (AUC) of
glucose concentrations during the entire 120 min of the 75-g oral glucose
tolerance test was calculated according to the trapezoid method as:
0.5 (0.5 c0 þ c30 þ c60 þ c90 þ 0.5 c120), where c is the concentration.
Other measurements were performed using the usual techniques of the
clinical laboratory.
Study of the effects of weight loss induced by bariatric surgery
Seven Caucasian morbidly obese (BMI ¼ 50.4
±
9.0 kg m
2
, age ¼ 40
±
10
years (mean
±
s.d.)) and normotolerant women were recruited. An oral
glucose tolerance test, an intravenous glucose tolerance test and a
euglycemic hyperinsulinemic clamp were randomly performed within
1 month before surgery and 1 month after surgery. The main procedures
were as previously described.
20
All subjects were nonsmokers and were
not receiving statins or antidiabetic medication. Patients with signs of
infection were excluded. The study protocol was approved by the
institutional ethics committee of the Catholic University of Rome (Rome,
Italy). The nature and purpose of the study were carefully explained to all
the subjects before they provided their written consent to participate.
Ex vivo studies in human adipose tissue
Explants of SC adipose tissue were obtained from five obese female
participants undergoing elective open abdominal surgery (gastrointestinal
bypass) after an overnight fast, cultured and treated as previously
described.
21
The mean age was 46
±
6.4 years (range, 39–58 years) and
the BMI 44.9
±
12.4 kg m
2
. None of the subjects had a history of hepatic
or renal disorders. The study had the approval of the ethics committee of
the participant institutions and all patients gave informed written consent.
Mice model experiments
Experimental protocols were conducted in accordance with the French
government policies and were validated by the local animal ethics
committee of the Rangueil Hospital and the Services ve
´
terinaires de la
Sante
´
et de la production animale, Ministe
`
re franc¸ais de lervices ve
´
te.
C57bl6 adult male mice were housed for less than 12 h of light/12 h of dark
cycle, in a temperature-controlled environment. Body weight and food
intake were measured weekly. In all the experiments, mice were killed after
overnight fasting. White mesenteric fat, taken as a reflection of visceral
adiposity, and/or SC adipose tissue samples were then removed, weighed
and either fixed in buffered formalin or snap frozen in liquid nitrogen. All
samples were stored at 80 1C until use.
p53 expression in mesenteric fat from GLP-1R KO mice and high-fat diet-fed
mice. Four- to five-week-old C57BL/6J male mice (Iffa-Credo, L’Arbresle,
France) were fed with normal chow (energy content: 12% fat, 28% protein
and 60% carbohydrate) or a high-fat carbohydrate-free diet (energy
content: 72% fat (corn oil and lard), 28% protein and o1% carbohydrate
22
)
for 4 weeks (acute stimuli) or 9 months (diet-induced obese mice). Notably,
this diet has been defined to promote a heterogeneous obesity status.
Hence, it is suitable to study the natural and diverse adaptation of mice
to metabolic stress, and to provide a heterogeneous population that can
be secondarily classified in obese and non-obese mice, as previously
described.
23
In vitro studies
Culture and differentiation of human adipocytes. Isolated pre-adipocytes
(Zen-Bio Inc., Research Triangle Park, NC, USA) were plated and
differentiated to MAs as previously described.
24
Pre-adipocytes and MAs
were harvested and stored at 80 1C for RNA extraction to study gene
expression levels during adipogenesis. The experiment was performed in
triplicate for each sample.
Effects of MCM and Rs on p53 gene expression. Adipocytes were incubated
with fresh media (control), fresh media containing macrophage-condi-
tioned medium (MCM, 5%) from THP-1 cells previously treated with
10 ng ml
1
lipopolysaccharides (LPS) or fresh media containing Rs (2 mM).
After 48 h, the supernatants were centrifuged at 400 g for 5 min, the cells
harvested, and pellets and supernatants were stored at 80 1C for RNA
and protein analysis.
The embryonic fibroblast mouse cell line 3T3-L1 (Zen-Bio Inc.) was
maintained in Dulbecco’s modified Eagle’s medium containing 20 m
M of
glucose, 10% fetal bovine serum, 100 units ml
1
penicillin and
100 mgml
1
streptomycin. Two days after confluence, insulin (5 mgml
1
),
dexamethasone (0.5 m
M) and IBMX (0.5 mM) mixture was added and
maintained for 2 days, followed by 5 days with a medium containing
insulin (5 mgml
1
). Cells were then considered MAs, harvested and stored
at 80 1C. In parallel, non-differentiated 3T3-L1 were maintained for
7 days in Dulbecco’s modified Eagle’s medium containing 10 (low),
20 (normal) and 200 (high) m
M of glucose, 10% fetal bovine serum,
100 units ml
1
penicillin and 100 mgml
1
streptomycin to analyze the
specific effect of glucose on p53 gene expression.
Gene expression analyses
RNA was prepared from both fat biopsy samples and cellular debris using
the RNeasy Lipid Tissue Mini Kit (Qiagen, Gaithersburg, MD, USA). The
integrity of each RNA sample was checked with an Agilent Bioanalyzer
(Agilent Technologies, Palo Alto, CA, USA). Total RNA was quantified by
means of the GeneQuant spectrophotometer (GE Health Care, Piscataway,
NJ, USA) and 3 mg of RNA was then reverse transcribed to cDNA using the
High Capacity cDNA Archive Kit (Applied Biosystems, Darmstadt, Germany)
according to the manufacturer’s protocol.
Gene expression was assessed by real-time PCR using the LightCycler
480 Real-Time PCR System (Roche Diagnostics, Barcelona, Spain), using
p53 in human adipose tissue
FJ Ortega et al
2
International Journal of Obesity (2013) 1 – 9 & 2013 Macmillan Publishers Limited
TaqMan technology suitable for relative gene expression quantification.
The reaction was performed following the manufacturer’s protocol in a
final volume of 7 ml. The cycle program consisted of an initial denaturing of
10 min at 95 1C, then 45 cycles of a 15-s denaturizing phase at 92 1C and a
1-min annealing and extension phase at 60 1C. Replicates and positive and
negative controls were included in all the reactions. The commercially
available and pre-validated TaqMan primer/probe sets used were as
follows: Cyclophilin A (PPIA, 4333763) was used as endogenous control
for all target genes in each reaction, and tumor-related protein 53
(p53, Hs01034249_m1), transformed mouse 3T3 cell double minute 2
(MDM2, Hs00234753_m1), solute carrier family 2 (facilitated glucose
transporter) member 4 (GLUT4, Hs00168966_m1), insulin receptor
substrate 1 (IRS1, Hs00178563_m1), leptin (LEP, Hs00174877_m1), fatty
acid synthase (FASN, Hs00188012_m1), tumor necrosis factor-alpha
(TNF-a, Hs01113624_g1), interleukin-6 (IL-6, Hs00985639_m1), adiponectin
(ADIPOQ, Hs00605917_m1) and the cluster differentiation molecules 14
(CD14, Hs02621496_s1) and 68 (CD68, Hs02836816_g1) were the target
genes. P53 (Mm01731287_m1), calgranulin A (s100a8, Mm00496696_g1),
lipopolysaccharide-binding protein (lbp, Mm00493139_m1) and fatty acid
binding protein 4 (fabp4, Mm00445878_m1) gene expression in mice was
assessed and expressed as a ratio relative to eukaryotic 18S rRNA
(18S, Hs99999901_s1). The second derivative maximum method was used
for the determination of the crossing points (Cps). A Cp value was obtained
for each amplification curve, and the DCp value was first calculated by
subtracting the Cp value for human PPIA or 18S from the Cp value for each
sample and transcript. Fold changes compared with the endogenous
control were then determined by calculating 2
Df
, so gene expression
results are expressed in all cases as an expression ratio relative to the
endogenous control expression, according to the manufacturer’s
instructions.
p53 protein analyses by ELISA
Trying to evaluate protein levels and the activation degree of p53 in
human adipose tissue (n ¼ 28; OM and SC paired fat samples), we used
three solid-phase sandwich enzyme-linked immunosorbent assays
(ELISAs) that detect endogenous levels of total p53 and phospho-p53
(Ser15) protein, and acetylated lysines in p53, respectively (Cell
Signaling, Izasa S.A., Barcelona, Spain). The analyses were performed
following the manufacturer’s instructions. After incubation with cell
lysates, total p53 protein, phospho-p53 (Ser15) and p53-acetylated
lysines were captured by the coated antibody in three independent
experiences. Following extensive washing procedures, mouse anti-
bodies were added to detect the captured total p 53 protein, phospho-
p53 (Ser15) and p53-acetylated lysines, respectively. Horseradish
peroxidase substrate-linked anti-mouse antibody was then used to
recognize the bound detection antibody. Horseradish peroxidase
substrate (3,3’,5,5’-tetramethylbenzidine base) was added to develop
color. The magnitude of absorbance for this developed color (450 nm)
was proportional to the quantity of total p53 protein, phospho-p53
(Ser15) and p53-acetylated lys ines, respectively.
Statistical analyses
Descriptive results of c ontinuous variable s are expressed as mean
±
s.d.
Before statistical analysis, normal distr ibution and homogeneity of the
variances were evaluated using Levene’s test. Variables were given a base
log
10
-transformation if necessary. These parameters were analyzed on a
log scale an d tested for significan ce on that scale. The anti-log-
transformed values of the means (geometric mean) are reported in the
following table. The relation between variables was tested using
Pearson’s test and stepwise multiple linear regression analysis. ANOVA
and the unpaired t-tests were used for comparisons of quantitative
variables. The general linear model was also used to calc ulate OM at p53
gene expression after adjusting for clinical variables. The statistical
analyses were performed using the program SPSS (version 13.0; SPSS Inc.,
Chicago, IL, USA).
RESULTS
Adipose p53 and obesity-associated inflammation
Gene expression of p53, MDM2, IRS1, GLUT4, LEP, TNF-a and CD68
was quantified in the SC and OM adipose tissue from a final
cohort of 230 subjects (76 paired samples). The anthropometric
and metabolic characteristics of the participants are summarized
in Table 1.
For the whole cohort, OM p53 was positively associated with
BMI (r ¼ 0.246, P ¼ 0.001, Figure 1a and Supplementary Figure S1a
in the online appendix) and percent fat mass (r ¼ 0.199, P ¼ 0.006,
Table 2). In contrast, SC p53 was not associated with BMI
(Figure 1b). No sex-related differences for these associations were
found. On the other hand, MDM2 (Supplementary Figure S1b)
gene expression levels were downregulated in adipose
from obese subjects (Table 1), concomitantly with GLUT4
(Supplementary Figure S1c) and IRS1 (Supplementary Figure
S1d). Both OM and SC MDM2 mRNA were associated with GLUT4
(r ¼ 0.343, Po0.0001 and r ¼ 0.307, P ¼ 0.012), IRS1 (r ¼ 0.391,
Po0.0001 and r ¼ 0.403, P ¼ 0.022) and the gene expression of
fatty acid synthase in OM and SC samples of adipose tissue
(r ¼ 0.331, Po0.0001 and r ¼ 0.432, P ¼ 0.014, respectively). Inter-
estingly, OM p53 expression also correlated with MDM2 (r ¼ 0.291,
P ¼ 0.008) in non-obese individuals (but not in obese subjects),
and with TNF-a (r ¼ 0.36, P
¼ 0.001) and CD68 (r ¼ 0.246, P ¼ 0.004)
gene expressions in OM adipose tissue (Table 2).
There is evidence indicating that mRNA may not necessarily
predict the translated protein levels. In this regard, measurements
of total p53 protein ( þ 11%, P ¼ 0.04, Supplementary Figure S2a),
phosphorylated (
Ser15
P)-p53 ( þ 16%, P ¼ 0.08; Supplementary
Figure S2b) and acetylated (A)-p53 ( þ 17%, P ¼ 0.01;
Supplementary Figure S2c) in human fat depots from a
subpopulation of 28 individuals (28 paired samples of OM and
SC adipose tissue) corroborated that the p53 levels in OM (but not
in SC fat) were increased with obesity. However, the activation
state of p53 (that is, the ratio
Ser15
P-p53/p53 and A-p53/p53) in
OM and SC human adipose tissue was not significantly related to
the parameters of obesity.
SC p53 expression was inversely related to cholesterol
(r ¼0.212, P ¼ 0.029) and low-density cholesterol (r ¼0.207,
P ¼ 0.041). The same relationship was noticed between OM p53
gene expression, circulating cholesterol (r ¼0.257, P ¼ 0.02) and
low-density cholesterol (r ¼0.3, P ¼ 0.01) in the subset of obese
individuals (n ¼ 115) (Table 2). Of note, OM (but not SC) MDM2 was
also associated with cholesterol (r ¼ 0.188, P ¼ 0.035) and low-
density lipids (r ¼ 0.204, P ¼ 0.026).
p53 gene expression in mesenteric adipose tissue from mice
models
In concordance with values of p53 in humans and concomitantly
with inflammatory markers such as s100a8 ( þ 124%, P ¼ 0.016),
lbp ( þ 125%, P ¼ 0.029) and fabp4 ( þ 79.5%, P ¼ 0.044), p53
mRNA was significantly increased in mesenteric fat from diet-
induced obese mice ( þ 57.6%, P ¼ 0.003) when compared with
lean mice (Figure 1c). Accordingly, p53 gene expression was
significantly increased ( þ 29.8%, P ¼ 0.004) in fat depots from
high-fat diet-fed mice when compared with normal chow diet-fed
mice (Figure 1d).
p53 during in vitro adipogenesis and isolated fat cells. The
monitoring of p53 gene expression and protein levels during
the adipogenic maturation of human pre-adipocytes and 3T3-L1
cells to MAs disclosed the decreased expression of p53 (Figure 2a
and Supplementary Figure S3a in the online appendix, respe-
ctively). Upon differentiation, the human pre-adipocytes developed
microscopically visible lipid droplets starting on the seventh
day. Concomitantly, there was a decrease in p53 expression
( 39.3%, P ¼ 0.038) accompanied by increased anti-inflammatory
mediators such as ADIPOQ (B107-fold,
Po0.0001) but decreased
pro-inflammatory cytokines such as IL-6 ( 97.5%, P ¼ 0.001).
MDM2 expressions succinctly increased with differentiation
(B2-fold, P ¼ 0.002).
In agreement with these results, the stromal-vascular cell
fraction was mainly responsible for p53 gene expression in both
p53 in human adipose tissue
FJ Ortega et al
3
& 2013 Macmillan Publishers Limited International Journal of Obesity (2013) 1 – 9
SC and OM fat depots, as p53 mRNA was significantly lower
( 43.3%, Po0.0001) in ex vivo isolated MAs (Figure 2b).
Effects of LPS-MCM and Rs on p53 gene expression in fully
differentiated adipocytes
As expected, LPS-induced macrophage-conditioned medium (5%)
administration increased LEP (Figure 2e) and IL-6 (B16-fold,
Po0.0001), lowering the expression of adipogenic and/or anti-
inflammatory factors such as ADIPOQ (Figure 2d). On the other
hand, Rs administration (2 mM) reduced LEP (Figure 2e) and IL-6
( 64%, Po0.0001), and increased the expression of ADIPOQ
(Figure 2d) as well as
p(panTyr)
IRS1/
total
IRS1 ratios (data not shown)
in MAs. In agreement with previous findings showing triggered
p53 expression by inflammation, LPS-induced macrophage-con-
ditioned medium treatment increased ( þ 35%, Po0.0001)
whereas Rs significantly decreased ( 40%, P ¼ 0.005) p53
gene expression in cultured human SC adipocytes (Figure 2c).
LPS by itself was able to induce the upregulation of p53 gene
expression also in mature 3T3-L1 adipocytes ( þ 93%, P ¼ 0.019;
Supplementary Figure S3b).
Adipose tissue p53 and altered glucose tolerance
OM (but not SC) p53 expression levels correlated with fasting
glucose (Figure 3a), with glucose concentrations at 0
0
(r ¼0.39,
P ¼ 0.036), 90
0
(r ¼0.4, P ¼ 0.035) and 120
0
(r ¼0.45,
P ¼ 0.016, n ¼ 28) during the oral glucose tolerance test, and
was significantly associated with the area under the curve for
glucose (r ¼0.39, P ¼ 0.039) in the random subsample of 28
subjects. Interestingly, OM p53 was strongly associated with
glycated hemoglobin (r ¼0.337, Po0.0001; Figure3b), a well
known measure of integrated glucose levels. Indeed, multiple
linear regression analyses to predict OM p53 levels revealed that
glycated hemoglobin (Po0.0001) and BMI (P ¼ 0.048) contributed
independently to explain 13.7% (Po0.0001) of OM p53 variance
(Table 2) after controlling for age. This association was of
special relevance among the non-obese subjects, and was not
Table 1. Anthropometrical and biochemical characteristics of study subjects
Whole cohort Non-obese without IGT Non-obese and IGT Obese without IGT Obese and IGT P (ANOVA)
N 95 21 61 53
Sex (% women) 61.7 71.4 83.3 77.4
Age (years) 49
±
14 56
±
10 44
±
12 47
±
12 0.002
BMI (kg m
2
) 25.0
±
3.1 27.5
±
1.8 42.6
±
6.7 43.0
±
5.7 o0.0001
% fat 31.7
±
7.1 37.5
±
6.6 54.1
±
8.7 54.6
±
9.2 o0.0001
SBP (mm Hg) 125.0
±
19.5 136.7
±
17.1 134.9
±
17.9 141.6
±
18.8 o0.0001
DBP (mm Hg) 77.7
±
13.0 78.7
±
11.3 78.8
±
10.5 81.8
±
11.1 0.421
Fasting glucose (mg dl
1
) 85.7
±
10.3 135.8
±
54.2 89.1
±
10.4 131.5
±
50.3 o0.0001
Insulin (mUml
1
) 11.2
±
6.8 15.0
±
7.7 13.7
±
9.1 19.2
±
13.5 0.005
HOMA-IR 2.39
±
1.54 4.72
±
2.24 2.97
±
1.87 5.70
±
4.09 o0.0001
HbA1c (%) 5.4
±
0.5 6.9
±
2.2 5.0
±
0.6 5.8
±
1.4 o0.0001
Total cholesterol (mg dl
1
) 202.9
±
38.3 209.2
±
44.7 188.6
±
28.8 197.2
±
37.5 0.072
HDL-cholesterol (mg dl
1
) 57.0
±
16.2 55.2
±
23.6 54.4
±
13.9 60.2
±
47.5 0.743
LDL-cholesterol (mg dl
1
) 123.3
±
30.2 120.4
±
43.3 114.8
±
28.2 114.6
±
36.2 0.367
Fasting triglycerides (mg dl
1
) 104.9
±
47.0 179.3
±
100.8 106.0
±
50.3 146.3
±
86.2 o0.0001
OM p53 gene expression (AU) 0.043
±
0.017 0.035
±
0.016 0.053
±
0.022 0.050
±
0.017 0.001
SC p53 gene expression (AU) 0.040
±
0.010 0.044
±
0.012 0.039
±
0.010 0.041
±
0.013 0.585
OM MDM2 gene expression (AU) 0.039
±
0.020 0.031
±
0.010 0.033
±
0.020 0.029
±
0.016 0.034
SC MDM2 gene expression (AU) 0.033
±
0.009 0.038
±
0.015 0.022
±
0.006 0.026
±
0.006 o0.0001
OM GLUT4 gene expression (AU) 0.043
±
0.035 0.020
±
0.018 0.024
±
0.022 0.017
±
0.010 o0.0001
SC GLUT4 gene expression (AU) 0.060
±
0.034 0.042
±
0.014 0.046
±
0.025 0.030
±
0.023 o0.0001
OM IRS1 gene expression (AU) 0.018
±
0.012 0.012
±
0.007 0.011
±
0.004 0.010
±
0.006 o0.0001
SC IRS1 gene expression (AU) 0.015
±
0.010 0.015
±
0.006 0.012
±
0.007 0.009
±
0.003 0.049
OM LEP gene expression (AU) 0.164
±
0.164 0.268
±
0.187 0.276
±
0.130 0.327
±
0.137 o0.0001
SC LEP gene expression (AU) 0.622
±
0.311 0.757
±
0.236 0.954
±
0.309 0.856
±
0.340 0.001
OM TNF-a gene expression (AU) 0.004
±
0.003 0.002
±
0.002 0.005
±
0.003 0.005
±
0.002 0.05
SC TNF-a gene expression (AU) 0.005
±
0.004 0.003
±
0.001 0.003
±
0.001 0.004
±
0.002 0.017
OM CD68 gene expression (AU) 0.163
±
0.102 0.146
±
0.096 0.179
±
0.076 0.23
±
0.113 0.011
SC CD68 gene expression (AU) 0.241
±
0.105 0.201
±
0.109 0.186
±
0.095 0.226
±
0.115 0.275
Subpopulation (A) Non-obese without IGT (B) Obese without IGT (C) Obese and IGT Student t (A) vs (B)
N 8911
OM p53 (OD) 0.099
±
0.006 0.110
±
0.013 0.105
±
0.015 0.044
SC p53 (OD) 0.110
±
0.008 0.112
±
0.008 0.109
±
0.008 0.572
OM P-p53 (OD) 0.106
±
0.015 0.123
±
0.021 0.110
±
0.017 0.078
SC P-p53 (OD) 0.111
±
0.012 0.121
±
0.018 0.114
±
0.005 0.248
OM A-p53 (OD) 0.374
±
0.035 0.439
±
0.051 0.420
±
0.030 0.009
SC A-p53 (OD) 0.415
±
0.074 0.476
±
0.074 0.483
±
0.081 0.156
Abbreviations: AU, arbitrary units; BMI, body mass index; CD68, cluster differentiation 68 molecule; DBP, diastolic blood pressure; GLUT4, glucose transporter
type 4; HbA1c, glycated hemoglobin; HDL, high-density lipoprotein; HOMA-IR, homeostasis model assessment of insulin resistance value, calculatedinall
subjects as (glucose (mmol l
1
) insulin (mU l
1
)/22.5); IGT, impaired glucose tolerance; IRS1, insulin receptor soluble 1; LDL, low-density lipoprotein;
LEP, leptin; MDM2, mouse double minute 2; OD, optical density; OM, omental fat; p53, tumor protein 53; SBP, systolic blood pressure; SC, subcutaneous fat;
TNF-a, tumor necrosis factor-alpha. Bold refers to significant differences between groups. ANOVA’s P-values depict significant variations among all groups.
Data are means
±
s.d. Non-obese, BMIo30 kg m
2
; obese, BMIX30 kg m
2
.
p53 in human adipose tissue
FJ Ortega et al
4
International Journal of Obesity (2013) 1 – 9 & 2013 Macmillan Publishers Limited
0.12
r=0.246
p=0.001
r=-0.05
p=0.596
0.10
SC p53 gene expression (AU)
OM p53 gene expression (AU)
0.08
0.06
0.04
0.02
0.00
20 30 40
Body Mass Index (Kg m
–2
)
Lean mice Obese mice NC-feed mice
#
*
5.0
4.0
3.0
3.0
2.0
2.0
Mesenteric p53 gene
expression (AU)
Mesenteric p53 gene
expression (AU)
1.0
1.0
1.5
2.5
0.0
0.0
0.5
HF-feed mice
50 60 20 30 40
Body Mass Index (Kg m
–2
)
50 60
0.12
0.10
0.08
0.06
0.04
0.02
0.00
Figure 1. Upper panels: Linear relationships between body mass index (kg m
2
) and omental (OM; a) and subcutaneous (SC; b) tumor protein
53 (p53) gene expression levels. Values for men are represented as empty circles (J), and women by empty diamonds (B). Lower panels:
Mean
±
2.0 s.e. for the mean of p53 gene expression in high-fat diet-induced obese and lean mice (c). The acute effect of high-fat diet on p53
gene expression levels in adipose tissue was also analyzed (d). *Po0.05 and
#
Po0.0001 for comparisons with the control group.
Table 2. Correlations and multiple linear regressions for tumor p53 gene expressions in OM fat depots and study variables
All subjects Non-obese (BMIo30 kg m
2
) Obesity (BMIX30 kg m
2
)
Correlations rP r P r P
Age (years) 0.137 0.058 0.109 0.276 0.092 0.39
BMI (kg m
2
) 0.246 0.001 0.08 0.425 0.016 0.881
Fat mass (%) 0.199 0.006 0.012 0.907 0.036 0.738
Fasting glucose (mg dl
1
) 0.111 0.151 0.244 0.017 0.084 0.478
HbA
1c
(%) 0.337 o0.0001 0.381 o0.0001 0.238 0.082
Total cholesterol (mg dl
1
) 0.094 0.214 0.107 0.3 0.257 0.02
HDL-cholesterol (mg dl
1
) 0.073 0.343 0.21 0.042 0.049 0.673
LDL-cholesterol (mg dl
1
) 0.139 0.072 0.072 0.493 0.3 0.01
Fasting triglycerides (mg dl
1
) 0.056 0.463 0.161 0.12 0.01 0.932
OM MDM2 gene expression (AU) 0.069 0.422 0.291 0.008 0.096 0.489
OM GLUT4 gene expression (AU) 0.144 0.093 0.289 0.008 0.292 0.03
OM IRS1 gene expression (AU) 0.2 0.017 0.339 0.003 0.27 0.026
OM LEP gene expression (AU) 0.159 0.072 0.034 0.754 0.025 0.882
OM TNF-a gene expression (AU) 0.36 0.001 0.508 0.002 0.286 0.07
OM CD68 gene expression (AU) 0.246 0.004 0.356 0.001 0.016 0.909
SC p53 gene expression (AU) 0.18 0.119 0.223 0.22 0.168 0.275
Multiple linear regression Beta P
Age (years) 0.022 0.798
BMI (kg m
2
) 0.157 0.048
HbA
1c
(%) 0.317 o0.0001
Adjusted R
2
13.7 (
P
o0.0001)
Abbreviations: AU, arbitrary units; BMI, body mass index; CD68, cluster differentiation 68 molec ule; GLUT4, glucose transporter type 4; HbA
1c
, glycated
hemoglobin; IRS1, insulin receptor soluble 1; LEP, leptin; MDM2, mouse double minute 2; OM, omental fat; p53, protein 53; SC, subcutaneous fat; TNF-a,
tumor necrosis factor-alpha. Beta is the standardized regression coefficient that allows evaluating the relative significance of each independent variable
in multiple linear regression analysis. Adjusted R
2
expresses the percentage of the variance explained by the independent variables in the different models
(i.e., 0.50 is 50%). Significant associations are indicated in bold.
p53 in human adipose tissue
FJ Ortega et al
5
& 2013 Macmillan Publishers Limited International Journal of Obesity (2013) 1 – 9
age-dependent (Table 2). Concurrently, p53 was positively
associated with the gene expression of insulin action biomarkers
in OM adipose tissue, such as GLUT4 and IRS1, especially among
non-obese participants (r ¼ 0.289, P ¼ 0.008 and r ¼ 0.339,
P ¼ 0.003, respectively; Table 2).
p53 gene expression in mesenteric adipose tissue from mice
models
Of note, GLP-1R KO mice, a genetic mouse model of glucose
intolerance,
25
showed decreased expressions of mesenteric p53
( 45.4%, P ¼ 0.017) when compared with wild-type mice
(Figure 3c). This was in agreement with the notion that impaired
glucose tolerance is associated with decreased p53 expression in
human OM adipose tissue.
Effects of surgery-induced weight loss on SC p53 gene expression
The characteristics of the subjects included in this longitudinal
study are shown in Supplementary Table S1. In an independent
cohort of morbidly obese women (n ¼ 7), bariatric surgery-
induced weight loss (60.1
±
11.7 vs 49.5
±
3.4 kg m
2
) led to
significantly increased SC p53 ( þ 44.9%, P ¼ 0.029; Supplementary
Figure S4a) gene expression levels despite the surgery-induced
downregulation of biomarkers of inflammation (I L-6, 89%,
P ¼ 0.036) and macrophage infiltration (CD14, 54.2%, P ¼ 0.02)
(Supplemental Table S1). On the other hand, i ncreased GLUT4
( þ 89.9%, P ¼ 0.01; Supplementary Figure S4c) and IRS1
( þ 99.1%, P ¼ 0.007; Supplementary Figure S4d) as well as
decreased LEP ( 55.3%, P ¼ 0.024; Supplementary Figure S4e)
expressions were concomitant to decreased weight and
increased insulin sensitivity (hyperinsulinemic euglycemic M value).
No significant changes for MDM2 gene expression were found
(Supplementary Figure S4b).
Effects of metformin on SC p53 gene expression
In the context of its well known enhanced AMP-activated protein
kinase effects, metformin (10 mmol l
1
) led to significantly
0.08
0.07
0.06
p53 gene
expression (AU)
p53 gene
expression (AU)
0.05
0.04
0.03
0.02
0.01
0.00
0.07
0.06
0.05
0.04
0.03
0.02
0.01
0.00
SVCs
*
Mature
adipocytes
#
Pre-
adipocytes
Adipocytes
(7th day)
Mature adipocytes
(14th day)
Control
0.0
2.0
4.0
6.0
8.0
10.0
*
#
+MCM (5%)
#
+Rs (2 uM)
Control
+MCM (5%)
+Rs (2 uM)
Control
+MCM (5%)
+Rs (2 uM)
*
*
*
0.0
0.5
p53 gene
expression (AU)
AdipoQ gene
expression (AU)
LEP gene
expression (AU)
1.0
1.5
4.0
3.0
2.0
1.0
0.0
Figure 2. Mean
±
2.0 s.e. for the mean of p53 gene expression at the 0th, 7th and 14th days after inducing adipogenesis (a), and in the cellular
fractions of adipose tissue (stromal-vascular cells and MAs) isolated from fresh human adipose tissue ( b). Lower panels represent the effect in
p53 (c), ADIPOQ (d) and LEP (e) gene expression of LPS-induced macrophage-conditioned medium and Rs in MAs. *Po0.05 and
#
Po0.0001 for
comparisons with pre-adipocytes/non-treated cells.
0.12
r=-0.111
p=0.151
r=-0.337
p <0.0001
0.10
0.08
OM p53 gene
expression (AU)
0.06
0.04
0.02
0.00
0.12
0.10
0.08
OM p53 gene
expression (AU)
0.06
0.04
0.02
0.00
60 80 100 120
140
Fasting glucose (mg dL
–1
) Hb1Ac (%)
NC-fed
WT mice
NC-fed
GLP-1R KO mice
456789
10
3.0
2.0
Mesenteric p53 gene
expression (AU)
1.0
*
1.5
2.5
0.0
0.5
Figure 3. Linear relationships between fasting glucose (a) and glycated hemoglobin (Hb1Ac, b) with p53 gene expression levels. Values for
men are represented as empty circles (J); and women as empty diamonds (B). Striped bars (c) represent mean
±
2.0 s.e. for the mean of p53
gene expression in mesenteric (Mes) adipose tissue from normal chow-fed GLP-1R KO transgenic and wild-type mice. *Po0.05 for
comparisons between group.
p53 in human adipose tissue
FJ Ortega et al
6
International Journal of Obesity (2013) 1 – 9 & 2013 Macmillan Publishers Limited
increased p53 gene expression levels ( þ 42%, P ¼ 0.018;
Supplementary Figure S5) ex vivo, in explants of human SC
adipose tissue.
Effects of glucose on p53 gene expression in 3T3-L1.Togain
insight into the potential underlyi ng molecular links between
increased glucose and p53 gene expressions in adipocytes, the
present study performed further experiments in 3T3-L1 pre-
adipocytes cultured with different concentrations of glucose
(low: 10; normal: 20 and high: 200 m
M). Interestingly, high
glucose concentrations significantly d ownregulated p53
( 27%, Po0.0001; Figure 4a), in parallel to the expression
pattern of biomarkers for insulin sensit ivity such as IRS1
( 44%, Po0.0001; Figure 4c) and AdipoQ ( 97%, P ¼ 0.005;
Figure 4e ).
DISCUSSION
The present work aimed at evaluating p53 in human adipose
tissue in association with obesity-associated inflammation
and insulin resistance. We found a dual relationship: the
upregulation of p53 linked to inflammation seemed to be
counterbalanced by the high glucose and insulin resistance-
dependent downregulation.
Inflammation-dependent effects on adipose tissue p53 gene
expression
Inflammation is well known to upregulate p53. Reactive oxygen
species (ROS)-induced p53 activation causes the NF-kB-depen-
dent induction of inflammatory cyt okines and cell d eath.
26
As
NF-kB induction occurs as a response to stress and p53 arrests
cells in G1/S (where DNA repair may be initiated ), the induction
of p53 by NF-kB could be a mechanism by which cells can
recover from stressors.
27
The upregulation of p53 in the OM
(but not in SC) adipose tissue with obesity, with its well known
chronic low-grade inflammatory activity,
4,6
could be interpreted
in this conte xt as further indicated by positive associations with
markers of inflammation (TNF-a) and macrophage infiltration
(CD68) in the OM fat depots. Then, the findings in humans
confirmed increased p53 expression in the adipose tissue of Ay
mice, a model in which increased inflammatory activity is
prominent. We here also r eplicated increased adipose tissue
p53 in high fat-fed mice. Thus, in obese adipose tissue and high
fat-fed mice increased inflammatory cytokines may account for
enhanced p53, whereas in differ entiated adipocytes increased
anti-inflammatory adipokines like adiponectin and the decreased
inflammation (which is necessary for adipogenesis) may explain the
in vitro p53 downregulation during differentiation.
Adipose tissue senescence was recently demonstrated in obese
subjects by the presence of shortened telomeres.
7
This may
increase the local production of pro-inflammatory molecules and
the upregulation of p53 in adipose tissue. In line with these
findings, p53 expression was higher in the most inflammatory
cellular counterpart of the adipose tissue: the stromal-vascular
fraction. Furthermore, LPS-macrophage-conditioned medium led
to raised p53 expression in MAs, as expected because cytokines
are known to activate p53 expression in concert with NF-kB.
28
On
the other hand, Rs, with its well known anti-inflammatory activity,
led to the downregulation of p53 in MAs (current findings).
Of note, MDM2 expression levels in adipose w ere positively
associated with the gene markers of lipogenesis (that is, FASN
and ADIPOQ) and insulin sensitivity (that is, IRS1 and GLUT4),
decreasing concomitantly with obesity. The MDM2 protein is
one of the most important p53-ubiquitin lig ases that induces
p53 polyubiquitination and degradation.
29
Although increased
p53 levels led to p 53-mediated cell death, MDM2 is requir ed t o
maintain p53 at low levels and to suppress i ts ability to induce
cell death in proliferating and qui escent cell s.
30
The opposite
expression pattern of MDM2 and p53 in OM fat depots, as
well as during adipogenesis, highlights its obesity-related
modulation.
High glucose and insulin resistance effects on adipose tissue p53
expression
p53 expression in OM adipose tissue was inversely associated with
glycated hemoglobin, a measure of integrated glucose levels. p53
gene expressions were also negatively linked to insulin resistance
and positively with the expression of insulin sensitivity-related
genes (GLUT4 and IRS1). Moreover, the gastric bypass bariatric
surgery, known as an effective approach for achieving weight
loss in obese patients,
31
improving and even completely solving
most obesity-associated complications, particularly insulin resistance
and type 2 diabetes 20, increased SC p53 expression, concomitantly
with increased insulin sensitivity and decreased inflammation (IL-6)
and macrophage infiltration (CD14). These findings were confirmed
in an animal model of glucose intolerance, the GLP-1R KO mice 25,
0.010
0.008
#
#
*
*
0.003
0.002
0.001
0.000
*
#
0.0E0
2.0E-5
1.5E-5
1.0E-5
5.0E-6
0.0E0
0.0000
adipoq gene
expression (AU)
0.0005
0.0010
0.0015
0.0020
irs1 gene
expression (AU)
glut4 gene
expression (AU)
Low
glucose
Normal
glucose
High
glucose
Low
glucose
Normal
glucose
High
glucose
Low
glucose
Normal
glucose
High
glucose
Low
glucose
Normal
glucose
High
glucose
Low
glucose
Normal
glucose
High
glucose
*
#
1.0E-4
2.0E-4
3.0E-4
4.0E-4
5.0E-4
il6 gene
expression (AU)
p53 gene
expression (AU)
6.0E-4
7.0E-4
0.006
0.004
0.002
0.000
Figure 4. Mean
±
2.0 s.e. for the mean of p53 (a), Il-6 (b), IRS1 (c), GLUT4 (d) and ADIPOQ (e) gene expression levels in 3T3-L1 pre-adipocytes
cultured under conditions of ‘low’ (10 m
M), ‘normal’ (20 mM) and ‘high’ glucose (200 mM). *Po0.05 and
#
Po0.0001 for comparisons with cells
cultured with ‘normal’ glucose concentration in the medium.
p53 in human adipose tissue
FJ Ortega et al
7
& 2013 Macmillan Publishers Limited International Journal of Obesity (2013) 1 – 9
that also showed decreased expressions of p53 in adipose tissue.
Furthermore, the relatively high glucose levels of in vitro adipocyte
differentiation led to decreased p53 levels despite increased IL-6
gene expression ( þ 200%, Po0.0001; Figure 4b). Thus, according
to our results the higher the glucose concentration, the lower the
p53 gene expression in adipocytes.
On the other hand, AMP-activated protein kinase constitutes a
major metabolic regulator of energy homeostasis that upregulates
p53, ensuring the arrest of the cel l c ycle in peri ods of glucose
deprivatio n.
32
Interestingly, metformin, known to activate
AMP-activated protein kinase, led to raised p53 gene
expression in adipose tissue. In line with t his effect,
metformin also led to p53 induction in parallel to Bcl-2
downregulation in melanoma cells, leading to cell cycle
arrest and apoptosis.
33
All these results suggest that hyperglycemia and/or insulin
resistance are associat ed with decreased apopt osis. Previous
studies have shown that hyperglycemia inhibited rat vascular
smooth muscle cell apoptosis in vi tro
34
and in patients with
diabetes.
35
When nondiabetic vascular smooth muscle cells
were preincubated with increasing concentrations of glucose
for 48 h, the y became resist ant to apoptosis, similar to the
diabetic cells.
35
The decrease in apoptosis in vascular smooth
muscle cells was re flected in an increasing artery-media
thicknessinpatientswithdiabetes.Thedecreasedapoptosis
with worsening of hyperglycemi a here found (at least at the
level o f adipose tissue p53 gene expression) might have clinical
implications. Type 2 diabetic patients are known to lose less
weight after dieting than nondiabe tic obe se sub jects.
36,37
Here
we postulate that the decreased p53 expression induced by
hyperglycemia might limit adipose tissue renewal and
resistance to weight loss.
In summary, factors linked to inflammation, such as weight gain,
aging and high-fat diet, among others, amplify p53 gene
expression, whereas hyperglycemia leads to decreased p53
expression in human adipose tissue. The specific involvement of
p53 mechanisms in these processes in human adipose tissue
should be investigated further.
CONFLICT OF INTEREST
The authors declare no conflict of interest.
ACKNOWLEDGEMENTS
We grea tly appreciate th e technical ass istan ce of Gerard Pardo, Ester Guerra,
Oscar Rovira and Roser Rodriguez (Unit of Diabetes, Endocrinology and Nutrition,
Institut d’Investigacio
´
Biome
`
dica de Girona; Hospital Universitari de Girona
Dr Jos ep Trueta). The work of all the members of the multidisciplinary obesity
team of the Clı
´
nica Universitaria de Navarra is also gratefully acknowledged. This
work was supported by research grants from the Ministerio de Educa cio
´
ny
Ciencia (SAF2011-00214) and the Instituto de Salud Carlos III (ISCIIIRETIC RD06,
CIBERObN). RB is a recipient of funding from the Agence Nationale de la
Recherche (Florinfl am and Flora dip programs ) as well as f rom L’Instit ute Nationa le
du Di abe
`
te.
AUTHOR CONTRIBUTIONS
All authors of this manuscript have directly participated in the execution and analysis
of the study and have approved the final version submitted. FJO designed the study,
analyzed the biochemical variables, performed the statistical analysis and wrote the
manuscript. JMM-N, DM and MS analyzed the biochemical variables. JIR-H obtained
the biopsies, the anthropometrical characteristics and the written consent of
volunteers. EL and RB were responsible for mice, and the mouse models of obesity
and high-fat diet. GM obtained subcutaneous fat samples from morbid obese
volunteers before and after bariatric surgery. WR, RB, FT, GF and GM provided
important intellectual content. AZ also carried out the conception of the study. JMF-R
carried out the conception and coordination of the study, and contributed to writing
the manuscript.
DISCLAIMER
The contents of this m anuscript have not been copyrighted or published
previously. There are no directly related manuscripts or abstracts, published or
unpublished, by on e or more a uthors of this manuscript. The contents of this
manuscript are not now under consideration for p ublication elsewhere. The
submitted manuscript nor any similar manuscript, in whole or in part, will n ot be
copyrighted, submi tted or published elsewhere while the Journal is under
consideration.
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