MEMORIAS TRABAJOS LIBRES Conferencia Científica Anual sobre Síndrome Metabólico 2015 Efecto comparativo de cuatro modelos de dieta con diferente cantidad y tipo de grasa sobre la disfunción del tejido adiposo en pacientes con síndrome metabólico en estado postprandial PhD María Eugenia Meneses*, PhD Antonio Camargo-García*, PhD Cristina Cruz-Teno*, PhD Yolanda Jiménez-Gómez**, PhD Pablo Pérez-Martínez*, PhD Javier Delgado-Lista*, PhD María del Mar Malagón-Poyato**, PhD Francisco Pérez-Jiménez*, PhD Helen Roche***, PhD José López-Miranda* * Unidad de Lípidos y Arteriosclerosis, Servicio de Medicina Interna, IMIBIC/Hospital Universitario Reina Sofía/Universidad de Córdoba, Córdoba, España y CIBER Fisiopatología Obesidad y Nutrición (CIBEROBN), Instituto de Salud Carlos III, Madrid, ** Departamento de Biología Celular, Fisiología e Inmunología. IMIBIC, (CIBEROBN).Universidad de Córdoba, España, *** Nutrigenomics Research Group, UCD Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Republic of Ireland

1. “Efecto comparativo de cuatro modelos de dieta con diferente cantidad y tipo de
grasa sobre la disfunción del tejido adiposo en pacientes con síndrome
metabólico en estado postprandial”
Dra. María Eugenia Meneses a, Dr. Antonio Camargo-García a, Dra. Cristina Cruz-Teno a, Dra. Yolanda Jiménez-
Gómez b, Dr. Pablo Pérez-Martínez a, Dr. Javier Delgado-Lista a, Dra. María del Mar Malagón-Poyato b, Dr. Francisco
Pérez-Jiménez a, Dra. Helen Roche c, Dr. José López-Miranda a.
a Unidad de Lípidos y Arteriosclerosis, Servicio de Medicina Interna, IMIBIC/Hospital Universitario Reina Sofía/Universidad de Córdoba,
Córdoba, España y CIBER Fisiopatología Obesidad y Nutrición (CIBEROBN), Instituto de Salud Carlos III, Madrid. b Departamento de
Biología Celular, Fisiología e Inmunología. IMIBIC, (CIBEROBN).Universidad de Córdoba, España. c Nutrigenomics Research Group,
UCD Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Republic of Ireland.

2. Metabolic syndrome is a cluster of risk factors for the development of cardiovascular diseases and type
II diabetes mellitus.
(NCEP-ATP III (National Cholesterol Education Program Panel III) Circulation 2002, 106:3143
Impaired
fasting glucose
≥100 mg/dl
Triglycerides
≥150 mg/dl
HDL Cholesterol
Male <40 mg/dl Females <50 mg/dl
Hypertension
130/85 (mmHg)
Abdominal obesity (Waist)
Males ≥102 cm Females ≥88 cm
Metabolic
Syndrome
Metabolic Syndrome

3. Adapted Nutrition Reviews 2007, Vol.65, n 6; Drug Discovery Today, 2010, Vol, 7, Issues 3-4
Adipose tissue dysfunction
Neutral
energybalance
Adipose tissue
expandability
Insulin resistance
&
Metabolic syndrome
Lipotoxicity
Excess of
nutrients
Excesslipid safelystored
Metabolic homeostasis
maintained
Adipose tissue
dysfunction
Chronic lipid loading
Adipocyte hypertrophy
Cellularstress
(ER stress)
Hypertrophy
Hyperplasia
TG start to be accumulated in
other organs “ectopic fat”
Obesity
Low grade inflammation
Insulin resistant
adipocytes
Disrupted adipokine profile

4. IRE1 (inositol-requiring 1), PERK (double-stranded RNA-dependent protein kinase (PKR)-like ER kinase), ATF6 (activating transcription factor 6,
(eIF2α) α-subunit of eukaryotic translation-initiation factor 2
Nature Vol 454, 24 July 2008
Excess of nutrients (FFA, TG, Glucose)
Excess of nutrients (FFA, TG, Glucose)
XBP-1
BiP
CRT
CNX
PDIA-3
GSTP1
Expression of endoplasmic reticulum stress-related genes in adipose tissue

5. The Journal of Clinical Investigation, December 2003, Volume 112, Number 12
Hypertrophy
Hyperplasia
Hypoxia
p65
IkBα
IκBβ
IL-6
MCP-1
IL1β
Expression of genes related with inflammatory response in adipose tissue.

6. Patients with Metabolic Syndrome are particulary vulnerable, since they show an exacerbated
hypertriglyceridemia response and abnormalities in the postprandial metabolism of lipoproteins.
Postprandial State
Expert Rev Cardiovasc Ther 2004;2:89-105, Adapted of www.lipidsonline.org
Stressful situation of the homeostasis by increase in lipid proinflammatory particles.

7. Objectives
1. Endoplasmic reticulum stress: XBP-1, CNX, CRT, PDIA-3, BiP, GSTP1.
2. Inflammatory response: p65, IkBα, IκBβ, IL-6, MCP-1, IL1β.
3. To analyze the relationship between adipose tissue gene expression and metabolic
risk indicators such as sensitivity to insulin, postprandial lipemia values and body
mass index.
To investigate the effect of the long-term consumption in fasting state and at the
postprandial state of four dietary models with different quantity and quality of fat on
the expression of genes related to:

8. 39 patients(25 females and 14 males) with metabolic syndrome were randomized to
receive one of four diets:
Postprandial study
AT Samples
12 weeks 4 hours
AT Samples AT Samples
n= 10
n= 8
n= 9
n= 12
n= 10
n= 8
n= 9
n= 12
Dietary intervention
High saturated fatty acids diet (HSFA)
38%E fat, 15%E protein, 47%E Carb
(16%E SFA;12%EMUFA; 6%E PUFA)
High monounsaturated fatty acids diet (HMUFA)
38 %E fat, 15%E protein, 47%E Carb
(8%E SFA; 20 %E MUFA; 6 %E PUFA)
Low-fat, high complex carb diet (LFHCC)
28 %E fat, 15%E protein, 57%E Carb
(8%E SFA; 11 %E MUFA; 6 %E PUFA)
Low-fat, high complex carb diet (LFHCC-n3)
28 %E fat, 15%E protein, 57%E Carb
(8%E SFA; 11 %E MUFA; 6 %E PUFA )
Total EPA and DHA, g/d :1.24
HSFAmeal
65%E fat, 10%E protein, 25%E Carb
(38% E SFA, 21% E MUFA 6% PUFA)
HMUFA meal
65%E fat, 10%E protein, 25%E Carb
(12% E SFA, 43% E MUFA, 10% E PUFA)
LFHCC meal
65%E fat, 10%E protein, 25%E Carb
(21% E SFA, 28% E MUFA, 16% E PUFA)
LFHCC-n3 meal
65%E, 10%E protein, 25%E Carb
(21%E SFA, 28%E MUFA, 16 %E PUFA)
(1.24g/dof LC n-3 PUFA)
Material and Methods

9. Subcutaneous adipose tissue samples collection
Subcutaneous adipose tissue samples were obtained from the abdominal region with instrument Bard® Magnum (ref.
MG1522), needles Bard® Magnum Core (ref. MN1410)
RNA isolation from adipose tissue
Gene expression by RT-PCR
The PCR reaction was carried out using the iQ SYBR Green kit (BioRad)
Thermal cycler system Mx3005
(Stratagene)
iQ5 iCycler
(BIO-RAD)
OpenArray ™ NT Cycler system
(Applied Biosystems)
Expression values were obtained as relative expression of the target gene versus the constitutively expressed gene
Adipokines
Inflammation related genes
ER related genes Lipid Metabolism related genes
Statistical analysis
SPSS Version 15. (Chicago, IL, USA). We performed ANOVA for repeated measurements and one-way
ANOVA in order to analyze statistical differences between diets. A study of the relationship among parameters was
also carried out using Pearson’s linear correlation coefficient.

10. P value corresponds to ANOVA statistical analysis.
Baseline characteristics of 39 patients with MetS (25 females and 14 males)
HSFA (n = 8) HMUFA (n = 9) LFHCC (n = 12) LFHCC n-3 (n = 10) P
1. Age (years) 57.8 ± 3.1 57.1 ± 2.3 56.5 ± 2 54.8 ± 2.1 0.839
2. BMI (kg/m2
) 36 ± 1.2 34.5 ± 1.2 35.7 ± 1 35 ± 1.2 0.817
3. Waist circumference 111.7 ± 3.1 104 ± 2 109 ± 3.1 108.3 ± 3.3 0.420
4. TC (mg/dl) 204 ± 19 192.2 ± 11.1 206.8 ± 14.9 196.9 ± 10 0.877
5. TG total (mg/dl) 226.7 ± 65.2 159.1 ± 20.9 161.5 ± 17.5 158.1 ± 20.3 0.422
6. LDL-c (mg/dl) 129.7 ± 13 135.9 ± 9.6 148.2 ± 12.1 140.4 ± 8.8 0.693
7. HDL-c (mg/dl) 41 ± 4.5 44.4 ± 3.3 43.4 ± 3.6 41.4 ± 2.9 0.906
8. Glucose (mg/dl) 117.7 ± 6.2 120.3 ± 7.1 106.1 ± 3.2 125.1 ± 13.5 0.371
9. Insulin (mU/ml) 15.3 ± 1.3 11.5 ± 1.3 12.6 ± 1.4 13.3 ± 1.7 0.400
Results

11. Expression of endoplasmic reticulum stress-related genes after 12 wks of dietary intervention
Fasting Post-intervention
Camargo A*, Meneses ME*, Mol. Nutr. Food Res. 2013, 57, 2166–2176

12. XBP1 CARL CANXP Diet: 0.482
P Time: <0.001
P Diet/Time: 0.678
0.00
0.05
0.10
0.15
0.20
0.25
HSFA HMUFA LFHCC LFHCC n-3
RelativeExpression
* * **
P Diet: 0.648
P Time: 0.821
P Diet/time: 0.548
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
HSFA HMUFA LFHCC LFHCC n-3
RelativeExpression
P Diet: 0.700
P Time: 0.192
P Diet/Time: 0.450
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.70
HSFA HMUFA LFHCC LFHCC n-3
RelativeExpression
PDIA3
BIP GSTP1
0.00
1.00
2.00
3.00
4.00
5.00
6.00
HSFA HMUFA LFHCC LFHCC n-3
RelativeExpression
P Diet: 0.186
P Time: 0.148
P Diet/Time: 0.490
0.00
1.00
2.00
3.00
4.00
5.00
6.00
HSFA HMUFA LFHCC LFHCC n-3
RelativeExpression
P Diet: 0.836
P Time: <0.001
P Diet/TIme: 0.893
* * *
0.00
1.00
2.00
3.00
4.00
HSFA HMUFA LFHCC LFHCC n-3
RelativeExpression
P Diet: 0.521
P Time: 0.108
P Diet/TIme: 0.670
Expression of endoplasmic reticulum stress-related genes at 4 hr after ingestion of the meal
Fasting postintervention Postprandial
Postprandial state
Camargo A*, Meneses ME*, Mol. Nutr. Food Res. 2013, 57, 2166–2176

13. Expression of genes related with inflammatory response after 12 wks of dietary intervention
Fasting Post-intervention
Meneses, et al. Mol. Nutr. Food Res. 2011, 55, 1759–1770

14. P Diet:0.758
P Time:0.101
P Diet/Time: 0.747
0.00
0.01
0.02
0.03
0.04
0.05
HSFA HMUFA LFHCC LFHCC n-3
RelativeExpression
P Diet:0.769
P Time: 0.003*
P Diet/TIme: 0759
* *
0.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
HSFA HMUFA LFHCC LFHCC n-3
RelativeExpression
P Diet:0.731
P Time: <0.001*
P Diet/Time: 0.841
* ***
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
0.50
HSFA HMUFA LFHCC LFHCC n-3
RelativeExpression
p65 IKBα IKBβ2
0
10
20
30
40
50
60
70
80
90
100
HSFA HMUFA LFHCC LFHCC n-3
RelativeExpression
P Diet: 0.820
P TIme: <0.001*
P Diet/Time: 0.800
*** *
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
HSFA HMUFA LFHCC LFHCC n-3
RelativeExpression
P Diet: 0.421
P Time: 0.001*
P Diet/TIme: 0.498
****
0.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
HSFA HMUFA LFHCC LFHCC n-3
RelativeExpression
n.d n.dn.dn.d
IL-6
P Diet: 0.766
MCP-1 IL1β
Expression of genes related with inflammatory response at 4 hr after ingestion of the meal
Fasting postintervention Postprandial
Postprandial state

15. Pearson’s linear correlation coefficient P<0.005
Postprandial State
p65 IkBα IkBb2 IL1β IL6 MCP1
XBP1
Pearson´s
Correlation
-0.127 0.688 0.306 0.465 0.624 0.378
P value 0.442 0.000 0.058 0.003 0.000 0.018
CRL
Pearson´s
Correlation
0.282 0.540 0.186 0.243 0.047 0.018
P value 0.082 0.000 0.256 0.135 0.775 0.911
CNX
Pearson´s
Correlation
0.352 0.614 0.440 0.418 0.233 0.359
P value 0.028 0.000 0.005 0.008 0.153 0.025
PDIA3
Pearson´s
Correlation
0.298 0.507 0.477 0.291 0.247 0.413
P value 0.065 0.001 0.002 0.072 0.129 0.009
BiP
Pearson´s
Correlation
0.079 0.758 0.589 0.648 0.277 0.428
P value 0.634 0.000 0.000 0.000 0.088 0.007
GSTP1
Pearson´s
Correlation
0.089 0.428 0.262 -0.031 0.144 0.240
P value 0.592 0.007 0.107 0.850 0.382 0.141
Endoplasmic
reticulum stress
Inflammatory
response
Relationship between gene expression of the pathways studied.

16. Third tercil
High gene
expression
13 patients
Second tercil
Middle gene
expression
13 patients
First tercil
Low gene
expression
13 patients
ID patient Gene Expression Tercil
983 4,39 1
961 7,82 1
947 8,51 1
946 17,45 1
960 21,93 1
962 22,64 1
971 28,33 1
967 30,78 1
951 33,83 1
980 37,92 1
975 39,41 1
958 40,36 1
956 40,46 1
964 40,95 2
977 41,36 2
944 41,45 2
949 42,81 2
966 43,56 2
985 44,18 2
963 45,96 2
972 45,96 2
945 51,08 2
941 53,57 2
965 53,83 2
986 54,41 2
943 58,36 2
978 61,26 3
981 68,69 3
959 73,55 3
955 75,56 3
969 88,62 3
957 93,54 3
976 93,63 3
970 96,31 3
950 99,60 3
984 102,74 3
982 148,68 3
953 225,79 3
968 281,40 3
13
13
13
Relationship between gene expression in adipose tissue and indicators
of metabolic risk. Analysis by tercils.
ID patient Gene expression
941 53,57
943 58,36
944 41,45
945 51,08
946 17,45
947 8,51
949 42,81
950 99,60
951 33,83
953 225,79
955 75,56
956 40,46
957 93,54
958 40,36
959 73,55
960 21,93
961 7,82
962 22,64
963 45,96
964 40,95
965 53,83
966 43,56
967 30,78
968 281,40
969 88,62
970 96,31
971 28,33
972 45,96
975 39,41
976 93,63
977 41,36
978 61,26
980 37,92
981 68,69
982 148,68
983 4,39
984 102,74
985 44,18
986 54,41

17. Third tercil
High gene
expression
13 patients
Second tercil
Middle gene
expression
13 patients
First tercil
Low gene
expression
13 patients
1st tercil 2nd tercil 3rd Tercil
Relationship between gene expression in adipose tissue and indicators
of metabolic risk.
P Tercil=0.006 P Time=<0.001 P Time/Tercil=0.022
130
150
170
190
210
230
250
0 1 2 3 4 5 6 8
CT(mg/dL)
Time h
TC vs. CANX tercils
P Tercil=0.012 P Time=0.006 P Time/Tercil=0.133
0
100
200
300
400
500
0 1 2 3 4 5 6 8
TG(mg/dL) Time h
TG vs. CANX tercils
b)a)
+
+
+
+
+
+
+
+
&
&
60
80
100
120
140
160
0 1 2 3 4 5 6 8
LDL-c(mg/dL)
Time h
LDL-c vs. XBP-1 tercils
P Tercil=0.043 P Time=0.200 P Time/Tercil=0.728
c)

18. Pearson’s linear correlation coefficient .
Relationship between gene expression in adipose tissue and indicators
of metabolic risk.
0
2
4
6
8
10
12
0,00 0,02 0,04 0,06 0,08 0,10 0,12 0,14 0,16
HOMA-IRIndex
p65 mRNA levels
r: 0.353
p=0.028
r: -0.377
p= 0.018
0
10
20
30
40
50
60
70
80
0,00 0,02 0,04 0,06 0,08 0,10 0,12 0,14 0,16
HDL(mmol/l)
p65 mRNA levels
r: 0.406
p=0.010
0
100
200
300
400
500
600
700
800
900
0,00 0,02 0,04 0,06 0,08 0,10 0,12 0,14 0,16
TG(mmol/l)
p65 mRNA levels

19. CONCLUSIONS
1. Our results suggest that an adequate postprandial response of ER stress in AT may be a
key factor that increases the effectiveness of TG clearance, and therefore may improve
metabolic flexibility.
2. Our results suggest that an exacerbated adipose tissue postprandial inflammatory
response occurs in individuals with MetS, which seems to be independent of the quality
and quantity of the dietary fat. This finding is important because the postprandial state is
probably the most common metabolic state that humans experience during the day, and
thus makes a substantial contribution to the environmental factors which increases
cardiometabolic risk.
3. Further research is needed to extend our knowledge about the effect of the type of diet on
postprandial inflammatory events in adipose tissue, the specific cellular and molecular
alterations, and the elucidation of nutritionally based therapeutic strategies for the MetS.