Prostaglandins, Leukotrienes and Essential Fatty Acids 102-103 (2015) 2129

Contents lists available at ScienceDirect

Prostaglandins, Leukotrienes and Essential

Fatty Acids
journal homepage: www.elsevier.com/locate/plefa

Deleterious effects of lard-enriched diet on tissues fatty acids

composition and hypothalamic insulin actions$
A.P.S. Dornellas a, R.L.H. Watanabe a, G.D. Pimentel a, V.T. Boldarine a, C.M.O. Nascimento a,
L.M. Oyama a, K. Ghebremeskel b, Y. Wang c, A.A. Bueno d, E.B. Ribeiro a,n
a

Department of Physiology, Division of Nutrition Physiology, Sao Paulo Federal University, Sao Paulo, Brazil
Lipidomics and Nutrition Research Centre, Faculty of Life Sciences and Computing, London Metropolitan University, London, United Kingdom
c
Department of Medicine, Division of Infectious Diseases, Section of Paediatrics, Institute of Reproductive and Developmental Biology, Imperial College
London, London, United Kingdom
d
Institute of Science and the Environment, University of Worcester, Worcester, United Kingdom
b

art ic l e i nf o

a b s t r a c t

Article history:
Received 21 December 2014
Received in revised form
28 August 2015
Accepted 1 October 2015

Altered tissue fatty acid (FA) composition may affect mechanisms involved in the control of energy
homeostasis, including central insulin actions. In rats fed either standard chow or a lard-enriched chow
(high in saturated/low in polyunsaturated FA, HS-LP) for eight weeks, we examined the FA composition
of blood, hypothalamus, liver, and retroperitoneal, epididymal and mesenteric adipose tissues. Insulininduced hypophagia and hypothalamic signaling were evaluated after intracerebroventricular insulin
injection.
HS-LP feeding increased saturated FA content in adipose tissues and serum while it decreased
polyunsaturated FA content of adipose tissues, serum, and liver. Hypothalamic C20:5n-3 and C20:3n-6
contents increased while monounsaturated FA content decreased. HS-LP rats showed hyperglycemia,
impaired insulin-induced hypophagia and hypothalamic insulin signaling.
The results showed that, upon HS-LP feeding, peripheral tissues underwent potentially deleterious
alterations in their FA composition, whist the hypothalamus was relatively preserved. However, hypothalamic insulin signaling and hypophagia were drastically impaired. These ndings suggest that
impairment of hypothalamic insulin actions by HS-LP feeding was not related to tissue FA composition.
& 2015 Elsevier Ltd. All rights reserved.

Keywords:
Fatty acid
Obesity
Diet
Insulin signaling
Adipose tissue
Hypothalamus

1. Introduction
The hypothalamus is one of the major structures of the central
nervous system involved in metabolic sensing and hormone
feedback. Appetite and energy expenditure are nely tuned by a
combination of central and peripheral signals, including acute
hormonal signals of hunger and satiety, as well as hormonal signals related to long-term control of adiposity [1,2]. Neural and
hormonal inputs act in anabolic and catabolic hypothalamic
effector systems, modulating food intake and energy expenditure
in response to physiological needs in energy metabolism.
In the hypothalamus, insulin binds to insulin receptors present
in areas involved in the control of food intake, including the arcuate nucleus and the ventromedial hypothalamus [35]. The initial

Funding: Brazilian Agencies FAPESP, CNPq and CAPES

Correspondence to: Universidade Federal de So Paulo, Departamento de
Fisiologia, Rua Botucatu, 862-2 andar, Vila Clementino, So Paulo, SP 04023-062,
Brazil. Tel./fax: 55 11 2758 2690.
E-mail address: eliane.beraldi@gmail.br (E.B. Ribeiro).
n

http://dx.doi.org/10.1016/j.plefa.2015.10.003
0952-3278/& 2015 Elsevier Ltd. All rights reserved.

steps of insulin signaling in hypothalamic nuclei are similar to

those in muscle and adipose tissues [6,7]. The insulin receptor (IR)
belongs to a family of growth factor receptors, with two transmembrane subunits expressing intrinsic tyrosine kinase activity
[8]. Circulating insulin binds to the extracellular IR subunits, triggering its autophosphorylation in tyrosine residues, and stimulating the enzymatic activity of transmembrane subunits [9].
Phosphorylated IR triggers a cascade of intracellular substrate
phosphorylation, such as IR substrates (IRS) 1 and 2 [10].
Phosphorylated IRSs bind to and activate phosphatidylinositol3-kinase (PI3-K), which in turn phosphorylates membrane phospholipids. Such actions are followed by activation of
phosphatidylinositol-dependent protein kinases 1 and 2, and
phosphorylation of protein kinase B (Akt) serine residues [8].
Activation of the PI3-K pathway in hypothalamic nuclei mediates
insulin-induced hypophagia, stimulating POMC neurons and
inhibiting AgRP neurons, among other actions [11,12].
Obesity has been associated with hypothalamic insulin resistance and hyperphagia [6,13]. Previous studies have shown that

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A.P.S. Dornellas et al. / Prostaglandins, Leukotrienes and Essential Fatty Acids 102-103 (2015) 2129

saturated fat intake impaired hypothalamic insulin signaling by

stimulation of endoplasmic reticulum stress [14], protein phosphatase activity [15] and pro-inammatory signaling [16].
Several studies have demonstrated the deleterious effects of
saturated fat rich diets on adipose tissue fatty acid composition
and endocrine function [1719]. As cell membrane phospholipids
are in intimate and functional contact with executive proteins, the
membrane fatty acid composition is decisive for tissue stability
and function [20,21]. Perturbations in tissue fatty acid composition
have been reported in a number of health conditions, such as
cardiovascular and metabolic diseases, cancer, neurodegenerative
and behavioral disorders [2225]. These perturbations are manifested mainly by increased saturated, trans and omega 6 fatty
acids, and concomitantly decreased omega 3 fatty acids [5,2628].
In this study, we investigated in male rats the effects of a lardenriched diet for 8 weeks in hypothalamus and peripheral tissue
fatty acid composition. Hypothalamic insulin-related signaling
pathways and insulin anorexigenic effects have also been
investigated.

2. Methods
2.1. Animals
This study was approved by the Sao Paulo Federal University
Research Ethics Committee, and all the procedures were carried out
in full compliance with its ethical guidelines. Fifty eight male Wistar
rats, weaned on the 21st day of life, were obtained from CEDEME
(Centro de Desenvolvimento de Modelos Experimentais para
Medicina e Biologia), and housed (four to ve rats per cage) in the
animal house of the Division of Nutrition Physiology, Sao Paulo
Federal University, under controlled lighting (12 h light/dark cycle,
lights on at 6:00) and temperature (2271 C), with free access to
standard rat chow and water. After two weeks acclimatization the
rats were randomised into two groups, and fed a standard rat chow
(Control) or lard-enriched chow (high in saturated/low in polyunsaturated FA, HS-LP) ad libitum for 8 weeks, as described below.

Table 1
Total energy calorie value (kcal/g), protein content, carbohydrates, alimentary ber,
mineral residues (g/100 g) and fatty acid composition (% of total fatty acids) of
control chow and lard-en riched chow.
Control chow

Lard-enriched chow

Energy (kcal/g)
Protein (g/100 g)
Carbohydrates (g/100 g)
Alimentary ber (g/100 g)
Mineral residues (g/100 g)
Total fat

2.7
22.4
39.1
11.4
11.9
4.8

4.1
23.6
26.8
15.1
9
22

Fatty acid composition (% total)

c14:0
c16:0
c18:0
SFA
ratio c16:0/c18:0

0.1
12.3
2.4
14.8
5.1

1
20.9
10.9
32.8
1.9

c16:1n7
c18:1n9
c18:1n7
c20:1
MUFA

0
24.6
1.1
0.2
25.9

1.7
36
2.3
0.6
40.5

c18:3n3
c18:2n6
PUFA
n-6/n-3

4.1
54.9
59
13.3

1.4
23.1
24.5
16.6

SFA/PUFA

0.3

1.3

sedation after an overnight fast. Trunk blood was collected, serum

immediately obtained by centrifugation and stored at  80 C until
analysis. Retroperitoneal (RET), epididymal (EPI) and mesenteric
(MES) fat pads, hypothalamus and liver were quickly dissected,
weighted and immediately stored at  80 C. Serum glucose was
determined by enzymatic colorimetric method (Glicose Pap
Liquiform, Labtest Diagnstica, Brazil) and insulin by radioimmunoassay (Rat insulin RIA kit, Millipore, USA).

2.2. Diets

2.5. Diet and tissue fatty acid composition

The diets were prepared in the food laboratory of the Division of

Nutrition Physiology, as described previously [29]. The standard rat
chow used in this study (Nuvilab CR1, Nuvital, Brazil) was ground
and enriched with lard, casein, sucrose and soybean oil, in the
proportions 50/18/20/10/2. Casein was added to obtain a protein/
energy ratio similar to the control diet, and soybean oil as a source
of essential fatty acids. Lukewarm water was added to achieve the
consistency necessary for perfect homogenization of the mixture,
subsequently passed through a milling machine for the production
of pellets, dried in a forced ventilation oven at 60 C for 24 h.
Samples of each diet were analysed in the laboratory of Bromatology and Microbiology of Foods, Sao Paulo Federal University, for
determination of macronutrients, and the diet fatty acid composition was determined by gas chromatography (Table 1).

Total lipids were extracted from the control and lard-enriched

diets, serum, hypothalamus, liver, RET, EPI and MES. Samples were
homogenized with hexane/isopropanol (3:2 v/v) containing 0.01%
butylated hydroxytoluene (BHT, Sigma, St Louis, MO), and washed
with chloroform/methanol/water (2:1:1 v/v/v). After low speed
centrifugation, the organic layer was obtained, evaporated to
dryness under a stream of oxygen-free nitrogen (OFN), and lipids
partitioned again in the chloroform layer of a chloroform/methanol/water solution (8/4/3 v/v/v). The resulting total lipid extracts
were dried under OFN, sealed in air-tight tubes and dispatched in
dry ice to the London Metropolitan University for fatty acid
analysis.
Fatty acid analysis was performed as standardized previously
[30]. Briey, fatty acid methyl esters (FAME) were obtained by
heating the samples with 15% acetyl chloride in dry methanol in a
sealed tube at 70 C for 3 h under OFN. The reaction was stopped
with the addition of 5% NaCl solution at room temperature, and
FAMEs were extracted by washing three times with petroleum
ether containing 0.01% BHT. FAMEs were separated by gas chromatography (HRGC MEGA 2 series, Fisons Instruments, Milan,
Italy) tted with a capillary column (BPX70, Thames Restek, UK).
Peak areas were quantied (EZChrom Chromatography Data System, Scientic Software Inc., San Ramon, CA, USA), and the values
presented represent the percentage of total fatty acids.

2.3. Food and energy intake

Twenty-four hours food intake and body weight were measured weekly. Energy intake was calculated based on weekly food
intake (g/100 g BW) and diet energy density (kcal/g).
2.4. Tissue weight, glucose and insulin blood levels
At the end of the 8 weeks dietary intervention period, a set of
rats from each group was sacriced by decapitation without

A.P.S. Dornellas et al. / Prostaglandins, Leukotrienes and Essential Fatty Acids 102-103 (2015) 2129

2.7. Evaluation of hypothalamic insulin signaling pathway by Western Blotting

One week after the experiment described above took place, rats
were deprived of food for 6 h, lightly anesthetized with ketamine/
xylazine intraperitoneally, and intracerebroventricularly injected
with 200 mU of insulin (Humulin, Eli Lilly, USA) or saline. Fifteen
minutes later, rats were decapitated, the hypothalamus quickly
removed and immediately homogenized in 1.0 mL of solubilization
buffer (10 mM EDTA, 100 mM Tris pH 7.5, 10 mM sodium pyrophosphate, 100 mM sodium uoride, 10 mM sodium orthovanadate, 2 mM PMSF, 2 g/mL aprotinin, 1% Triton X-100). Insoluble
material was removed by centrifugation (40 min, 14000 rpm, 4 C).
Supernatant protein concentration was determined colorimetrically (BCA Protein Assay, Bioagency Biotecnologia, Brazil).
Tissue extracts (100 mg) were denatured in boiling water for 5 min
in Laemmli buffer [33] containing 200 mM DTT.
Protein extracts were separated by SDS-PAGE, transferred onto
nitrocellulose membrane and incubated with primary antibody
anti-IR- (sc-711), p-IR- (sc-25103), IRS-1 (sc-559) (Santa Cruz
Biotechnologies, USA), IRS-2 (3089), Akt (9272), p-Akt (9271) (Cell
Signaling, USA). For evaluation of protein loading, all membranes
were stripped and reblotted with anti--tubulin primary antibody
(sc-58667). After incubation with the appropriate secondary
antibody conjugated with horseradish peroxidase, membranes
were developed by chemiluminescence. Quantitative analysis was
performed by densitometry using Scion Image software (Scion
Corporation, USA).

3.2. Fat pad mass and serum glucose and insulin

The HS-LP group showed highly signicant increases in RET, EPI
and MES masses (p o0.001), higher serum glucose (p o0.05) and
similar insulin (Table 2).
3.3. Fatty acids prole of diets and tissues
3.3.1. Diets
The lard-enriched diet contained more than double the amount
of total SFA (32.8%) as compared to the control diet (14.8%), and
1.5 times higher amount of total MUFA (40.5%) as compared to
Control (25.9%). Also, the lard diet contained only 33% of the
c18:3n-3 (Control 4.1%; HS-LP 1.4%) and 42% of the c18:2n-6
(Control 54.9%; HS-LP 23.1%) content as compared to Control. The
ratio n-6/n-3 is approximately 27% higher in HS-LP (Control 13.3;
HS-LP 16.6) (Table 1).
3.3.2. Hypothalamus
After 8 weeks of lard-enriched diet consumption, the HS-LP
hypothalami showed decreased amount of total MUFAs (p o0.05)
but increased ratio of C18:0/C18:1 (p o0.05), an indirect index of
enhanced activity of the microsomal enzyme stearyl-CoA desaturase (delta 9 desaturase). Higher levels of C20:3n-6 (po 0.01)
and lower C22:5n-6 (p o0.001) were also found in HS-LP rats.
Control

*
***

**

**

**

**

**

0
1

4
Weeks

40

2.8. Statistical analysis

30
20
10
0
1

4
Weeks

500
Body weight (g)

Results are expressed as mean and standard error. Comparisons

between groups (Control vs. HS-LP) were assessed using independent samples Student's t tests, equal variances assumed.
Phosphorylation data differences were assessed by one-way
ANOVA followed by Tukey's HSD (Honestly Signicant Difference) post-hoc tests for multiple comparisons. Statistical signicance was set at p o0.05.

HS-LP

12
Food intake
(g/100g/24h)

The second set of Control and HS-LP rats were anesthetized

with ketamine/xylazine (6.7/1.3 mg/100 g BW intraperitoneally),
and stereotaxically implanted with a 21-gauge guide cannula
aimed at the left lateral ventricle (from bregma: 0.9 mm anterior, 1.5 mm lateral, and  3.0 mm ventral) [31], as standardized
in our laboratory [32]. Animals were individually caged thereafter
with free access to their allocated chow and water. Four days after
surgery, correct cannula placement was conrmed by a positive
drinking response after a 20 ng angiotensin II intracerebroventricular injection. One week after surgery, C and HS-LP rats
received a 2 mL intracerebroventricular injection of 20 mU insulin
(Humulin, Eli Lilly, USA) or saline after 6 h fasting. Injections were
performed immediately before lights went off, and a known
amount of their respective chow was offered. The chow consumed
was quantied after 12 and 24 h.

food intake (g/100 g BW) was signicantly lower in HS-LP

(p o0.05 week 1, p o0.01 week 2 and thereafter), energy intake
was statistically similar between groups in week 2 and thereafter,
and body weight was signicantly higher in HS-LP (p o0.05 week
2 and 3, p o0.01 week 4 and thereafter) (Fig. 1).

Energy intake
(Kcal/100g/24h)

2.6. Intracerebroventricular insulin injection and food intake

measurement

23

400

*#

300

*#

*#

*#

#
***

*#

#
***

200
100
1

3. Results
3.1. Food and energy intake and body weight
60-day old male Wistar rats were treated for 8 weeks with a
standard rat chow (Control) or a lard-enriched diet (HS-LP). During the course of the 8 weeks treatment, weekly-measured 24 h

Weeks

Fig. 1. Food (g/100 g BW/24 h, upper panel) and energy (kcal/100 g BW/24 h,
middle panel) intake and body weight (g, lower panel) of male rats treated with a
standard rodent chow (2.7 kcal/g, 15% energy from fat) or lard-enriched chow
(4.1 kcal/g, 52% energy from fat) for 8 weeks. Error bars indicate standard error of
the mean. N 20 for each group.
n
p o0.05 HS-LP vs. Control; nnp o0.01 HS-LP vs. Control; nnnp o 0.001 HS-LP vs.
Control.

24

A.P.S. Dornellas et al. / Prostaglandins, Leukotrienes and Essential Fatty Acids 102-103 (2015) 2129

Higher levels of C20:5n-3 (p 0.015), and a tendency to increased

C22:6n-3 (p 0.08) were found in HS-LP rats (Table 3).
3.3.3. Serum
Lower C14:0 (p o0.01) and C16:0 (p o0.05) were found in the
HS-LP group, followed by substantially higher C18:0 (p o0.001)
and total SFA (p o0.001). The ratio C16:0/C18:0 is signicantly
decreased in HS-LP (p o0.001), which could be attributed to a
reection of dietary provision or increased elongase activity. The
HS-LP treatment decreased C16:1n-7 (po 0.05) and C18:1n-7
(p o0.001), but did not change other MUFAs nor the total MUFA
content; however, as in hypothalamus it increased the ratio C18:0/
C18:1 (p o0.01). Lower amounts of C18:3n-3 (p o0.001), C20:5n-3
Table 2
Fat pad weight (g/100 g BW) and serum glucose (mg/dL) and insulin (ng/mL) of
male rats treated with a standard rodent chow (Control, 2.7 kcal/g, 15% energy from
fat) or lard-enriched chow (HS-LP, 4.1 kcal/g, 52% energy from fat) for 8 weeks. Data
are presented as means 7 SEM, n 14 for Control; n916 for HS-LP.

Fat pad (g/100 g)

Retroperitoneal
Epididymal
Mesenteric

Serum glucose (mg/dL)

Serum insulin (ng/mL)
n

Control

HS-LP

0.717 0.04
0.82 7 0.05
0.707 0.04
104.77 3.7
0.8 7 0.1

1.40 70.09nnn
1.47 70.10nnn
1.30 70.08nnn
119.6 76.3*
1.0 70.2

p o 0.05 HS-LP vs. Control.

p o0.001 HS-LP vs. Control.

nnn

(p o0.01), C22:5n-3 (p o0.01) and total n-3 PUFA (p o0.01) were

found in the HS-LP group. The HS-LP treatment lowered the
amounts of C18:2n-6 (p o0.001) and C20:3n-6 (po0.05), but
increased C20:4n-6 (po 0.01) and C22:5n-6 (p o0.001); the total
n-6 PUFA remained signicantly decreased (p o0.01). The total
PUFA content (n-6 n-3) was decreased (p o0.01), the ratio n-6/n3 was increased (p o0.01) and the ratio SFA/PUFA was increased
(p o0.001) (Table 3).
3.3.4. Liver
A signicant decrease in C24:0 (p o0.01) was found in the HSLP group, but this fatty acids accounts for less than 0.5% of total
fatty acids; no signicant differences were found in other SFAs.
Higher C18:1n-9 (p o0.01), C20:1 (p o0.05) and total MUFA
(p o0.01) were found in the HS-LP group, followed by lower
C18:1n-7 (p o0.001). As opposed to serum and hypothalamus, the
ratio C18:0/C18:1 was not signicantly higher in HS-LP. All the n-3
fatty acids quantied were signicantly reduced (po 0.05) in the
liver of HS-LP rats, but only C20:5n-3 did not reach statistical
signicance (p 0.39) despite an approximate 30% decreased
amount. The HS-LP treatment induced a more complex variation
in the n-6 fatty acid amounts: C18:2n-6 (p o0.05), C20:4n-6
(p o0.05) and the total n-6 PUFA (p o0.01) were lower, but
C18:3n-6 (p o0.01), C22:4n-6 (p o0.01) and C22:5n-6 (p o0.01)
were higher. C20:3n-6 (the C20:4n-6 precursor) was similar
between HS-LP and C. The total PUFA content (n-6 n-3) was
lower (po 0.01), the ratio n-6/n-3 (p o0.05) and SFA/PUFA
(p o0.001) were higher in HS-LP rats (Table 3).

Table 3
Fatty acid composition of liver, serum and hypothalamus total lipid extract of male Wistar rats treated with a standard rodent chow (Control, 2.7 kcal/g, 15% energy from fat)
or lard-enriched chow (HS-LP, 4.1 kcal/g, 52% energy from fat) for 8 weeks. Data are presented as means 7 SEM of total FAs (%). n 5 for each group.
Fatty acid

Total fatty acids (%)

Liver

C14:0
C16:0
C18:0
C24:0
SFA
C16:0/C18:0
C16:1n-7
C18:1n-9
C18:1n-7
C20:1
C24:1
MUFA
C18:0/C18:1
C18:3n-3
C20:5n-3
C22:5n-3
C22:6n-3
n-3
C18:2n-6
C18:3n-6
C20:3n-6
C20:4n-6
C22:4n-6
C22:5n-6
n-6
PUFA
n-6/n-3
SFA/PUFA

Serum

Control

HS-LP

0.3 7 0.04
17.6 7 0.48
14.7 7 0.44
0.5 7 0.02
337 0.51
1.2 7 0.06
0.7 7 0.15
7.3 7 0.25
3.3 7 0.17
0.2 7 0.02
0.2 7 0.01
11.7 7 0.43
1.4 7 0.07
0.3 7 0.03
0.3 7 0.02
1.5 7 0.11
57 0.49
7.17 0.37
187 0.74
0.3 7 0.01
0.6 7 0.02
25.17 0.39
0.9 7 0.03
0.2 7 0.05
457 0.7
52.17 0.75
6.4 7 0.35
0.6 7 0.02

0.2 7 0.03
16.8 7 0.65
16.17 1.56
0.3 7 0.04
33.4 7 0.93
1.17 0.12
0.4 7 0.04
17.5 7 1.87
27 0.13
0.3 7 0.04
0.17 0.02
20.3 7 2.05
0.9 7 0.23
0.2 7 0.03
0.2 7 0.07
1.17 0.08
3.7 7 0.16
5.2 7 0.21
15.5 7 0.44
0.4 7 0.03
0.5 7 0.02
20.17 1.7
1.5 7 0.15
0.9 7 0.17
38.9 7 1.14
44.17 1.31
7.5 7 0.17
0.8 7 0.01

nn

nn
nnn
n

nn

n
n
nn
n
nn

n
nn
nn
nn
nn
n
nnn

Hypothalamus

Control

HS-LP

Control

HS-LP

0.4 70.02
20.4 70.41
12 70.26
0.2 70.02
32.9 70.46
1.7 70.06
0.8 70.12
9.5 70.44
2 70.14
0.1 70.03
0.1 70.01
12.6 70.5
1 70.05
0.4 70.02
0.5 70.05
0.9 70.05
2.5 70.22
4.3 70.18
20.5 70.37
0.2 70.01
0.3 70.03
26.1 70.4
0.6 70.06
0.1 70.03
47.9 70.66
52.3 70.72
11.1 70.5
0.6 70.02

0.3 7 0.02
197 0.39
18.3 7 0.48
0.17 0.01
37.7 7 0.69
17 0.03
0.5 7 0.03
10.9 7 0.71
1.17 0.02
0.2 7 0.02
0.17 0.04
12.7 7 0.75
1.6 7 0.12
0.17 0.02
0.2 7 0.03
0.6 7 0.04
2.4 7 0.14
3.2 7 0.12
11.5 7 0.59
0.2 7 0.03
0.3 7 0.02
31.4 7 1.33
0.6 7 0.11
0.5 7 0.11
44.4 7 0.67
47.6 7 0.71
13.7 7 0.53
0.8 7 0.02

nn

0.17 0.01
19.9 70.35
18.17 0.2
0.3 7 0.04
38.4 7 0.46
1.1 70.02
0.5 7 0.02
20.9 7 0.22
47 0.12
1.1 70.05
0.05 7 0.01
26.5 7 0.37
0.7 7 0.01
0.047 0.02
0.077 0.01
1.08 7 0.31
137 0.78
14.2 70.89
1.4 7 0.42
ND
0.2 7 0.01
10.7 70.43
3.4 7 0.07
0.4 7 0.01
16.17 0.12
30.3 7 1.01
1.2 7 0.07
1.3 7 0.06

0.17 0.01
207 0.2
187 0.16
0.3 70.02
38.4 70.27
1.17 0.01
0.4 70.02
20.2 70.15
3.6 70.09
1.17 0.05
0.05 70.01
25.4 70.2
0.8 70.01
0.05 70.01
0.137 0.01
0.63 70.08
14.7 7 0.33
15.5 7 0.36
1.3 7 0.19
ND
0.3 70.01
11.17 0.18
3.4 70.06
0.3 70.01
16.4 7 0.24
31.9 7 0.51
1.17 0.02
1.2 7 0.03

n
nnn

nnn
nnn
n

nnn

nn
nnn
nn
nn

nn
nnn

n
nn

nnn
nn
nn
nn
nnn

n
n

nn

nn

nnn

SFA: saturated fatty acids; MUFA: monounsaturated fatty acids; PUFA: polyunsaturated fatty acids; DHA: docosahexaenoic acid, EPA: eicosapentaenoic acid, ND: not detected.
n

p o0.05.
p o0.01.
nnn
p o 0.001.
nn

A.P.S. Dornellas et al. / Prostaglandins, Leukotrienes and Essential Fatty Acids 102-103 (2015) 2129

25

Table 4
Fatty acid composition of retroperitoneal, epididymal and mesenteric white adipose tissue total lipid extract of male Wistar rats treated with a standard rodent chow
(Control, 2.7 Kcal/g, 15% energy from fat) or lard-enriched chow (HS-LP, 4.1 Kcal/g, 52% energy from fat) for 8 weeks. Data are presented as means 7 SEM of total FAs (%). n
5 for each group.
Total fatty acids (%)
Fatty acid

C14:0
C16:0
C18:0
C24:0
SFA
C16:0/C18:0
C16:1n-7
C18:1n-9
C18:1n-7
C20:1
C24:1
MUFA
C18:0/C18:1
C18:3n-3
C20:5n-3
C22:5n-3
C22:6n-3
n-3
C18:2n-6
C18:3n-6
C20:3n-6
C20:4n-6
C22:4n-6
C22:5n-6
n-6
PUFA
n-6/n-3
SFA/PUFA

Retroperitoneal

Epididymal

Control

HS-LP

0.8 70.05
20.3 70.31
2.7 70.14
ND
23.9 70.37
7.5 7 0.42
2.8 70.5
24.27 0.2
2.7 70.12
0.2 70.01
ND
29.9 70.68
0.17 0.01
2.4 70.06
0.09 70.02
0.3 70.04
0.337 0.03
3.2 70.11
39.17 0.68
0.117 0.01
0.2 70.02
1.6 7 0.12
0.4 70.03
0.09 70.01
41.5 70.67
44.6 7 0.75
13.1 70.35
0.5 70.01

0.9 7 0.04
22 70.23
5.9 7 0.23
ND
28.8 7 0.12
3.7 7 0.17
2.4 7 0.16
39.8 7 0.24
2.8 7 0.03
0.3 7 0.01
ND
45.3 7 0.22
0.147 0.01
0.9 7 0.01
0.06 70.03
0.17 0.01
0.08 7 0.02
1.17 0.05
22.9 7 0.17
0.04 70.01
0.077 0.01
0.5 7 0.08
0.17 0.03
0.04 70.01
23.7 7 0.09
24.77 0.12
21.9 70.89
1.2 70.01

nn
nnn

nnn
nnn

nnn

nnn

nnn
nn
nnn

nnn
nnn
nnn
nnn
nnn
nnn
nnn
nnn
n
nnn
nnn
nnn
nnn

Mesenteric

Control

HS-LP

0.8 7 0.03
20.8 7 0.23
37 0.04
ND
24.67 0.21
77 0.14
2.17 0.08
257 0.3
2.7 7 0.15
0.2 7 0.01
ND
307 0.41
0.117 0
2.3 7 0.08
0.067 0.001
0.2 7 0.02
0.167 0.02
2.7 7 0.1
39.7 7 0.44
0.08 7 0.01
0.147 0.01
17 0.09
0.3 7 0.03
0.067 0.01
41.2 7 0.43
43.9 7 0.49
15.3 70.47
0.6 7 0.01

0.8 70.03
21.8 7 0.12
6.3 70.05
ND
28.9 70.13
3.5 70.04
1.7 7 0.1
41.4 70.3
2.8 70.05
0.4 70.02
ND
46.4 70.19
0.14 70.001
0.7 70.03
0.05 70.02
0.17 0.001
0.05 70.01
0.9 70.04
22.4 70.21
0.02 70.01
0.08 70.01
0.3 70.04
0.17 0.03
0.04 7 0.01
22.9 70.17
23.8 70.2
26.9 71.16
1.2 7 0.01

nn
nnn

nnn
nnn
n
nnn

nnn

nnn
nnn
nnn

nnn
nnn
nnn
nnn
nnn
nn
nnn
nn

nnn
nnn
nnn
nnn

Control

HS-LP

0.6 7 0.04
19.3 7 0.39
3.2 7 0.09
ND
23.2 7 0.45
67 0.18
1.3 7 0.12
25.9 7 0.3
2.7 7 0.1
0.2 7 0.01
ND
30.1 70.45
0.117 0
27 0.11
0.05 7 0.001
0.2 7 0.02
0.157 0.01
2.4 7 0.1
41.5 7 0.61
0.077 0.01
0.117 0.01
0.8 7 0.03
0.3 7 0.02
0.05 7 0.01
42.8 7 0.61
45.1 70.69
18.2 70.59
0.5 7 0.02

0.7 7 0.02
20.2 7 0.12
7.8 70.21
ND
28.8 7 0.19
2.6 7 0.08
1.2 70.02
42.2 7 0.16
2.9 7 0.04
0.5 7 0.01
ND
46.8 7 0.17
0.177 0.01
0.6 7 0.01
0.03 7 0.01
0.17 0.001
0.05 7 0.01
0.7 7 0.02
21.9 7 0.11
0.017 0
0.05 7 0.01
0.3 7 0.02
0.17 0.02
0.02 7 0.01
22.4 7 0.11
23.17 0.13
30 70.81
1.2 70.01

n
nnn

nnn
nnn

nnn

nnn

nnn
nnn
nnn

nnn
nnn
nnn
nnn
nnn
nn
nnn
nnn

nnn
nnn
nnn
nnn

RET: retroperitoneal white adipose tissue; EPI: epididymal white adipose tissue; MES: mesenteric white adipose tissue; DHA: docosahexaenoic acid; EPA: eicosapentaenoic
acid; SFA: saturated fatty acids; MUFA: monounsaturated fatty acids; PUFA: polyunsaturated fatty acids; ND: not detected. Data are expressed as mean 7 SEM.
n

po 0.05.
p o0.01.
nnn
p o 0.001.
nn

3.3.5. Retroperitoneal, epididymal and mesenteric fat pads

The three white fat pads analysed were dramatically affected by
the HS-LP diet, and showed very similar patterns of variation
(Table 4). C16:0 (po0.05 at least), C18:0 (p o0.001) and total SFA
(p o0.001) were higher in HS-LP, and the ratio C16:0/C18:0 was
lower (p o0.001). C16:1n-7 was lower (p o0.05) in HS-LP RET
only, but C18:1n-9 (p o0.001), C20:1 (po 0.001), total MUFA
(p o0.001) and the ratio C18:0/C18:1 (p o0.01 at least) were
higher in all three HS-LP fat pads. C18:3n-3, C22:5n-3, C22:6n-3
and total n-3 PUFA were lower (p o0.001) in all HS-LP fat pads. No
signicant differences were found in C20:5n-3, despite an
approximate 33% reduction in EPI, 17% in RET and 40% in MES.
C18:2n-6 (po 0.001), C18:3n-6 (p o0.001), C20:3n-6 (p o0.01 at
least), C20:4n-6 (p o0.001) and C22:4n-6 (p o0.01 at least) were
lower in HS-LP fat pads; C22:5n-6 was lower only in EPI (p o0.05).
The total n-6 PUFA (po 0.001), total PUFA (p o0.001), ratio n-6/n3 (p o0.001) and ratio SFA/PUFA (po 0.001) were lower in the HSLP group.

in Control, an overall 19% reduction, but no signicant effect was

found in HS-LP (Fig. 2).
3.5. Hypothalamic insulin signaling pathway
IR protein levels were signicantly reduced (po 0.05) in HS-LP
(Fig 3A). IR tyrosine phosphorylation (pIR) was signicantly
increased after insulin injection in Control (po 0.05), a 143%
increase; however, this effect was not observed in HS-LP, who
showed a statistically insignicant, 14% increase (Fig. 3B). IRS-1
and IRS-2 protein levels were similar between groups (data not
shown). Akt protein levels were signicantly lower in HS-LP
(p o0.05) (Fig 3C). Akt serine phosphorylation was signicantly
increased after insulin injection in Control (49% increase, po 0.01)
but the same effect was not observed in HS-LP, who showed a
statistically insignicant, 15% increase (Fig. 3D).

3.4. Insulin-induced hypophagia

4. Discussion

Control rats showed signicantly reduced food intake (p o0.05)

in the rst 12 h after intracerebroventricularly injected insulin, as
compared to saline injection. However, insulin injection showed
no effect in the HS-LP group. On the second 12 h period, the
appetite-supressing effect of insulin had subsided in Control, and
no retarded effect was observed in HS-LP. The cumulative 24 h
intake was signicantly decreased (p o0.05) after insulin injection

Obesity is a well characterized public health issue and its correlation with other health conditions is clear. Enlarged adipose
mass leads to increased peripheral vascular resistance, macrophage inltration, metabolic syndrome and mild chronic inammatory conditions [3436]. Chronic positive energy balance is the
main causal factor for the development of obesity, but often the
abundant intake of affordable, palatable, energy-dense foods

26

A.P.S. Dornellas et al. / Prostaglandins, Leukotrienes and Essential Fatty Acids 102-103 (2015) 2129

energy from fat, 14.8% of saturated fatty acids). These results

conrm previous studies describing the effects of high fat diets as
inducers of obesity in animal models and humans [17,3739], and
the obesogenic effects of saturated fat [40,41]. Several molecular
mechanisms have been associated with increased adiposity
induced by high fat diets, including reduced rate of dietary fat
oxidation, higher lipogenesis and lower lipolysis rates, increased
uptake of circulating lipids due to higher lipoprotein lipase activity
[4244], and others.
In this study, HS-LP rats showed lower food intake, but similar
energy intake, as compared to the control group, from the second
week of treatment onwards. Previous studies have shown rats
treated with high fat diets showed increased body fat content and
decreased food intake [43,45]. It has also been shown the consumption of diets rich in saturated fat reduces the expression of
the orexigenic peptides neuropeptide Y [46] and agouti-related
protein [47] in the arcuate nucleus of hypothalamus, and increases
the secretion of the anorexigenic cholecystokinin [48].
After 8 weeks of dietary treatment, HS-LP rats showed
increased fasting glycaemia alongside normoinsulinemia, which
suggests the development of peripheral insulin resistance, in
agreement with previous studies [41,49]. The type of fat appears to
be decisive for the development of insulin resistance: mice treated
with either MUFA or SFA rich diets showed increased body mass,
but only those treated with SFA developed glucose intolerance
[50]. In corroboration with the latter, mice treated for 60 days with

triggers a nutritional imbalance that may be correlated with

metabolic disorders.
In the present study, male Wistar rats treated for 8 weeks with
a lard-enriched diet (HS-LP, 52% energy derived from fat, 32.8% of
saturated fatty acids) showed increased body and adipose tissue
masses, as compared to rats fed a standard chow (Control, 15%
30
#

Food intake (g)

24
18

12
6
0
Control HS-LP
- +
- +

Control HS-LP

0-12

12-24

group

Control HS-LP
- +
- +

insulin

0-24

hours

Fig. 2. Food intake during the rst and second 12-h periods, and during the entire
24 h period after intracerebroventricular injection of vehicle (solid bars) or insulin
(striped bars) of male rats treated with a standard rodent chow (2.7 kcal/g, 15%
energy from fat) or lard-enriched chow (4.1 kcal/g, 52% energy from fat) for
8 weeks. Error bars indicate standard error of the mean. Control, n 2328; HS-LP,
n 1921.
#
p o 0.05 vs. respective vehicle-treated group.

Control
Control

HS-LP

-tubulin

150

100
50

pIR/-tubullin

-tubulin

300
200
100
0

0
Control

+
Control

HS-LP

Control
Control

HS-LP

Akt

pAkt

-tubulin

-tubulin

150
100

50
0
Control

HS-LP

pAkt/-tubulin

Akt/-tubulin

IR

IR

IR/-tubullin

HS-LP

200

+
HS-LP

Insulin

HS-LP

##

150
100
50
0
+
Control

+
HS-LP

Insulin

Fig. 3. Top panels (A and B): hypothalamic insulin receptor (IR) protein levels (a) and tyrosine phosphorylation (b) of Control (Control, n 78) and High-saturated/low-PUFA
(HS-LP, n 89) groups. Bottom panels (C and D): hypothalamic Akt protein levels (a) and serine phosphorylation (b) of Control (n 68) and HS-LP (n 67) groups. Rats
were treated with intracerebroventricularly injected vehicle (  ) or insulin ( ). Error bars indicate standard error of the mean. np o 0.05 HS-LP vs. Control; #p o0.05 vs. the
respective saline-treated group; ##p o 0.001 vs. the respective saline-treated group.

A.P.S. Dornellas et al. / Prostaglandins, Leukotrienes and Essential Fatty Acids 102-103 (2015) 2129

a lard-enriched diet showed decreased adiponectin serum levels

[28], an adipokine directly related to insulin sensitivity and glucose homeostasis.
The HS-LP chow used in this study provided more than doubled
the amount of total SFA as compared to the Control chow. The ratio
of n-6/n-3 was 17:1 in the HS-LP diet, and 13:1 in the Control diet,
but the HS-LP diet contained only 33% of the total n-3, and only
42% of the total n-6, present in the Control diet. As expected, tissue
fatty acid composition has been dramatically changed in HS-LP
rats. Previous studies have shown the inuence of dietary fats in
the fatty acid composition of several tissues, including skeletal
muscle, subcutaneous fat, liver, heart, pancreas, stomach, intestine
and plasma [51], as well as on the development of metabolic and
brain disorders [23,52].
The fatty acid composition of the HS-LP white fat pads analysed
in this study showed dramatic variation in relation to Control: the
amounts of n-6 PUFAs were signicantly decreased, replaced by
substantially higher amounts of SFAs and MUFAs. The SFA geometrical conguration increases membrane phospholipid rigidity
and lipid bilayer tension, which impairs GLUT4 vesicle exteriorization, resulting in glucose intolerance [22,53]. Diabetes-induced
glucotoxicity contributes to -cell failure, and exposing cultured
human pancreatic islets to MUFAs reduced apoptosis induced by
high glucose and palmitic acid levels [54]. Additionally, it has been
described a butter-enriched diet had a detrimental impact on the
indirectly measured activity of stearoyl-Coa desaturase 1, which
correlated with metabolic disorders and obesity [55].
The liver fatty acid composition of HS-LP showed no changes in
C16:0 or C18:0 levels, but increased C18:1n-9, despite the chronic
intake of a high SFA-low PUFA diet. It has been described C18:0
presented high desaturation rate to its monounsaturated equivalent C18:1 in cultured hepatocytes [56], and the activity of the
Delta9-desaturase in liver was elevated by C14:0 treatment [57].
Even though we did not quantify the activity of the Delta9desaturase in the present study, we would speculate an
increased activity in liver tissue, as the excess of dietary SFA would
be desaturated into MUFAs and a considerable amount being
exported to other tissues for incorporation as phospholipids or
triglycerides.
The mild alteration of hypothalamic fatty acids composition
contrasts with the profound changes observed in the peripheral
tissues of the HS-LP rats. In a previous study, we observed a very
low content of trans fatty acids in the brain of 21-day and 90-dayold rats born from mothers fed with trans fatty acids during
pregnancy and lactation [26]. Other authors have reported a preferential incorporation of these fatty acids in liver and fat, with
protection of the brain tissue [58]. It can be hypothesized that a
similar mechanism accounted for the present observations but
further experiments are necessary to clarify this suggestion.
In addition to the above, the hypothalamus showed a small but
signicant decrease in total MUFA, despite higher MUFA levels in
the diet. Previous studies have found an inverse correlation
between cerebral spinal uid and whole blood C18:1n-9 in
humans [59,60]. Interestingly, a literature review claried shifting
a SFA-rich diet to a MUFA-rich diet improved insulin sensitivity,
whereas a moderate supplementation with n-3 PUFA did not have
the same level of effect on insulin actions [21]. In a rodent model
of obesity, saturated fat feeding induced hypothalamic cell apoptosis by activation of Toll-like receptor 4, a member of the IL-1/Toll
receptor family playing important roles in host defence and
inammatory processes [61].
We found increased C20:5n-3 (the C22:6n-3 precursor) and
C20:3n-6 (the C20:4n-6 precursor) in HS-LP total hypothalamus
lipid extract. Both C22:6n-3 and C20:4n-6 remained unchanged in
HS-LP, as well as the total n-3 and total n-6 PUFAs. C22:6n-3 and
C20:4n-6 are the main fatty acids in the medial basal

27

hypothalamus of cows [62], and the main fatty acids in major

neural systems in humans [63,64]. We hypothesize increased
C20:5n-3 and C20:3n-6, followed by unchanged C22:6n-3 and
C20:4n-6 in HS-LP hypothalamus is the result of a protective
mechanism in which peripheral tissues supply these essential fatty
acids for neural uptake, as evidenced by their concomitantly
reduced levels in peripheral tissues.
Impaired insulin-mediated signaling actions in neural systems
directly impacts on the development of metabolic diseases. The
anorexigenic effects of insulin are decisive for the maintenance of
body energy homeostasis, and decient insulin signaling has been
associated with obesity in various experimental models [6,49,65].
Moreover, hypothalamic resistance to insulin induced by a high
energy palatable diet preceded adipose tissue hypertrophy [66],
and the lack of Tumor Necrosis Factor Receptor had a protective
effect on reduced thermogenesis and impaired insulin signaling
induced by a high-fat diet [67]. In the present study, we examined
whether a 8 weeks lard-enriched dietary intervention affected
food intake after insulin intracerebroventricular administration;
insulin signicantly reduced food intake in the control rats, but no
actions were observed in the HS-LP ones, in agreement with
previous research [68,69].
Decreased hypothalamic IR protein levels we found in HS-LP
rats in our study; other studies however, employing different
experimental models and looking into gene expression instead,
have found either increased [49] or unchanged [68] IR levels.
Previous research from our group showed unchanged IR protein
levels in rats treated with soybean or sh oil enriched diets, as
compared to standard chow fed rats. [4] Even though we did not
investigate gene expression, but the amount of protein instead, it
is plausible to suggest that the lack of essential fatty acids
impaired the synthesis, translocation and or exteriorization of IR
protein.
In this study, HS-LP rats did not show signicantly increased IR
tyrosine phosphorylation after stimulation with intracerebroventricularly injected insulin, as seen in the control rats (Fig. 3B). A
previous study suggested increased IR serine phosphorylation may
be an important factor in reducing IR tyrosine phosphorylation [6].
We did not nd any differences in hypothalamic levels of IRS-1
and IRS-2 (data not shown). It has been shown obese insulinresistant Zucker rats presented moderately decreased IRS-1 and
IRS-2 hypothalamic expression [6]. On the other hand, it has also
been shown that consumption of a saturated fat rich diet for
1 week increased IR, IRS-1 and 2 gene expression in rat hypothalamus, but such perturbations subsided after 6 weeks on that diet,
suggesting the hypothesis of an adaptive mechanism [49].
Protein kinase B (Akt), the more distal insulin-signaling cascade
protein investigated in this study, is regulated by several molecular
mechanisms, and plays a key role in relevant biological processes
such as apoptosis, cell proliferation and differentiation [70,71],
glucose uptake in peripheral tissues [72,73], and activation of
hypothalamic anorexigenic neurons [74,75]. In this study, we
found lowered Akt protein levels in HS-LP (Fig 3C), increased Akt
serine phosphorylation in Control after intracerebroventricularly
injected insulin, and no effect in HS-LP (Fig. 3D). We therefore
propose the imbalance in fatty acid tissue composition is responsible for such impairment in HS-LP. In agreement with our proposition, previous studies have shown that insulin is less effective
in inducing satiety and Akt phosphorylation in the hypothalamus
of rats fed with saturated fat-rich diets [69,76]. Hypothalamus
inammation induced by saturated fat-rich diet has been dened
as a relevant factor in the impairment of insulin signaling [77].
The results presented in this study support previous reports of
central insulin resistance after dietary manipulations leading to
increased body adiposity. Impaired insulin-induced hypophagia
and impaired hypothalamic insulin signaling have been observed

28

A.P.S. Dornellas et al. / Prostaglandins, Leukotrienes and Essential Fatty Acids 102-103 (2015) 2129

in adult rats submitted to intrauterine food restriction, in rats fed a

trans fatty acid-enriched, and in rats fed a soybean oil-enriched
diet [4,26,70].
In summary, despite showing similar energy intake, male Wistar
rats treated with a high SFA-low PUFA diet for 8 weeks showed higher
body mass, adiposity and glycaemia as compared to Control rats. All
peripheral tissues analysed in this study showed dramatic perturbations in their fatty acid composition, showing signicantly higher SFA
and lower n-3 and n-6 PUFAs. However, the hypothalamus was not as
much affected, showing levels of total PUFAs similar to the control
group. HS-LP rats did not show the same level of insulin-induced
hypophagia as the controls, which is likely to be explained by
impaired insulin signaling. HS-LP rats also showed reduced levels of IR
phosphorylation.
In conclusion, the present ndings showed that the HS-LP diet
induced a preferential modication of fatty acids composition of
serum, liver and fat, while the hypothalamus had only minor
alterations.

Authors' contribution
APSD, RLHW and AAB designed the research, conducted
laboratory work, analysed the results and wrote the paper. GDP,
VTB and YW conducted laboratory work, analysed the results and
wrote the paper. CMON and LMO designed the research, analysed
the results and wrote the paper. EBR is the Principal Investigator,
designed the research, analysed the results and wrote the paper.
All authors commented on the nal versions of the paper, and
share primary responsibility for its nal content.

Acknowledgments
This study was supported by Grants from the Sao Paulo
Research Foundation (FAPESP, Brazil) (Grant no. 2007/05360-4),
the National Council for Scientic and Technological Development
(CNPq, Brazil) (Grant no. 309405/2013-0), and the Coordination for
the Improvement of Higher Education Personnel (Capes, Brazil).

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