A high oleic sunflower oil fatty acid esters of plant sterols mixed with dietary diacylglycerol reduces plasma insulin and body fat accumulation in Psammomys obesus.

Abstract

Metabolic syndrome is associated with subsequent development of cardiovascular diseases and type 2 diabetes. It is characterized by reduced response to insulin, central obesity, and dyslipidemia. Intake of plant sterols (PS) has been shown to confer a healthier lipid profile and ameliorate cardiovascular disease risk factors in experimental animals and humans. In this study we used an animal model of type 2 diabetes to assess the effects of a preparation of PS esterified to high oleic sunflower oil fatty acids mixed with dietary diacylglycerol (PS-HOSO) on diabetic related metabolic parameters. Psammomys obesus (P. obesus) were fed high energy (HE) diet supplemented by either PS-HOSO or control oil. Following 4.5 weeks of intervention, animals were divided into fasting and non-fasting modes prior to outcome measurements. Glucose and insulin levels as well as blood lipid profile, body weight, and fat accumulation were evaluated in fasting and non-fasting modes.
P. obesus fed with a HE diet displayed a characteristic heterogeneity in their blood glucose and insulin levels with a subset group displaying type 2 diabetes symptoms. PS-HOSO treatment significantly reduced total cholesterol (24%, P < 0.001) and non-HDL cholesterol (34%, P < 0.01) compared to the control diet. Among fasting animals, body weight at end point and epididymal fat-to-liver weight ratio were significantly (P < 0.05 each) reduced (7% and 16%, respectively) compared to controls. Interestingly, fasting blood glucose levels were similar between groups, whereas plasma insulin level at end point was 44% lower in the PS-HOSO group compared to control group (P < 0.0001)
PS-HOSO supplementation to diabetes-prone gerbils counteracts the increase in body weight and epididymal fat accumulation, and also results in a drop in circulating insulin levels. These effects are pointing out that PS-HOSO may serve as a functional ingredient for metabolic syndrome or diabetic sufferers, which not only influences body weight, but also prevents or reverses insulin resistance and hyperlipidemia.

Full text

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BioMed Central
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Lipids in Health and Disease
Open Access
Research
A high oleic sunflower oil fatty acid esters of plant sterols mixed
with dietary diacylglycerol reduces plasma insulin and body fat
accumulation in Psammomys obesus
Ehud Ziv*1, Natan Patlas1, Rony Kalman2, Dori Pelled3,4, Yael Herzog3,
Tali Dror3 and Tzafra Cohen3
Address: 1Diabetes Research Unit, Hadassah Medical Center and The Hebrew University, Jerusalem 91120, Israel, 2Animal facility, Hebrew
University, Jerusalem, Israel, 3R&D Department, Enzymotec LTD, Migdal-HaEmeq, Israel and 4Current address: Medical Department, NiTi Surgical
solutions, Netanya, Israel
Email: Ehud Ziv* - ehud@hadassah.org.il; Natan Patlas - patlas@hadassah.org.il; Rony Kalman - ronyk@savion.huji.ac.il;
Dori Pelled - dori@nitisurgical.com; Yael Herzog - yael@enzymotec.com; Tali Dror - talid@enzymotec.com;
Tzafra Cohen - Tzafra@enzymotec.com
* Corresponding author
Abstract
Background: Metabolic syndrome is associated with subsequent development of cardiovascular
diseases and type 2 diabetes. It is characterized by reduced response to insulin, central obesity, and
dyslipidemia. Intake of plant sterols (PS) has been shown to confer a healthier lipid profile and
ameliorate cardiovascular disease risk factors in experimental animals and humans. In this study we
used an animal model of type 2 diabetes to assess the effects of a preparation of PS esterified to
high oleic sunflower oil fatty acids mixed with dietary diacylglycerol (PS-HOSO) on diabetic related
metabolic parameters. Psammomys obesus (P. obesus) were fed high energy (HE) diet supplemented
by either PS-HOSO or control oil. Following 4.5 weeks of intervention, animals were divided into
fasting and non-fasting modes prior to outcome measurements. Glucose and insulin levels as well
as blood lipid profile, body weight, and fat accumulation were evaluated in fasting and non-fasting
modes.
Results: P. obesus fed with a HE diet displayed a characteristic heterogeneity in their blood glucose
and insulin levels with a subset group displaying type 2 diabetes symptoms. PS-HOSO treatment
significantly reduced total cholesterol (24%, P < 0.001) and non-HDL cholesterol (34%, P < 0.01)
compared to the control diet. Among fasting animals, body weight at end point and epididymal fat-
to-liver weight ratio were significantly (P < 0.05 each) reduced (7% and 16%, respectively)
compared to controls. Interestingly, fasting blood glucose levels were similar between groups,
whereas plasma insulin level at end point was 44% lower in the PS-HOSO group compared to
control group (P < 0.0001)
Conclusion: PS-HOSO supplementation to diabetes-prone gerbils counteracts the increase in
body weight and epididymal fat accumulation, and also results in a drop in circulating insulin levels.
These effects are pointing out that PS-HOSO may serve as a functional ingredient for metabolic
syndrome or diabetic sufferers, which not only influences body weight, but also prevents or
reverses insulin resistance and hyperlipidemia.
Published: 12 October 2009
Lipids in Health and Disease 2009, 8:42doi:10.1186/1476-511X-8-42
Received: 7 September 2009
Accepted: 12 October 2009
This article is available from: http://www.lipidworld.com/content/8/1/42
© 2009 Ziv et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Background
Metabolic syndrome represents a cluster of metabolic dis-
turbances that attained almost epidemic proportions
worldwide and is associated with subsequent develop-
ment of type 2 diabetes mellitus and cardiovascular dis-
ease [1,2]. It is characterized by central obesity [3,4],
hypertension, hyperglycemia and reduced response to
insulin (insulin resistance). In addition, metabolic syn-
drome sufferers display various degrees of dyslipidemia.
The main lipid metabolism abnormalities seen in these
subjects are increased plasma triglycerides (TG) and
reduced high-density lipoprotein cholesterol (HDL-c) lev-
els, along with a predominance of small low-density lipo-
protein (LDL) particles [5]. It has therefore been proposed
that alteration of the fat metabolism is a critical factor that
may precipitate the etiology of the disease, at least in some
cases. Favourable modifications of the lipid profile in suf-
ferers of metabolic syndrome or type-2 diabetes, may be
achieved by pharmaceutical means [6,7]. Recently, dietary
supplements and functional foods have been increasingly
used as lipid-lowering agents [8,9]. Intake of the n-9
monounsaturated fatty acid, oleic acid, was shown to be
inversely correlated with ischemic heart disease presuma-
bly due to hypolipidemic effects [10]. This notion was fur-
ther confirmed in several clinical intervention trials [11-
13]. Plant sterols (PS) were also reported to have signifi-
cant cholesterol-lowering properties [14]. Accordingly, PS
Intake decreased circulating cholesterol concentrations
via the suppression of intestinal absorption due to the
higher affinity of PS to micelles [15,16]. Recent works
have shown that PS incorporated into food matrices such
as margarines and spreads or in the form of dietary sup-
plements are bioactive for long term treatment periods
[17,18].
Israeli sand rat Psammomys obesus, (P. obsesus), a desert ger-
bil species has been extensively used as a genetically het-
erogeneous model for type 2 diabetes. P. obesus closely
resembles some human populations in which a subset of
the population goes on to develop diabetes when fed a
high energy diet [19,20]. The course of the disease devel-
opment in these animals can be divided into four phases:
Phase A, normoinsulinemia and normoglycemia; phase
B, hyperinsulinemia and normoglycemia; phase C, overt
diabetes, characterized by hyperinsulinemia and hyperg-
lycemia; and phase D, hypoinsulinemia and severe hyper-
glycemia as a result of β-cell degranulation, and markedly
reduced pancreatic insulin content [21]. As in humans,
obesity and increase in body fat underlie the development
of type 2 diabetes.
We have previously shown the efficacy of preparations of
PS esterified to high oleic oil fatty acids in reducing total
cholesterol (TC) levels in experimental animals [22] and
humans [23]. In addition, the preparations were shown to
reduce plasma lipid peroxidation.
In the present study, we investigated the effect of PS ester-
ified to high oleic sunflower oil (HOSO) fatty acids mixed
with dietary diacylglycerol (DAG) in HOSO matrix (PS-
HOSO) or soybean oil on the development of metabolic
syndrome and diabetic symptoms in P. obesus.
Methods
Animals and diets
Sixty male Psammomys obesus gerbils were obtained from
Harlan Laboratories Ltd (Jerusalem, Israel). After wean-
ing, the animals were maintained on a low-energy diet
containing 2.38 kcal/g (Koffolk, Petach Tikva, Israel) by
Harlan Laboratories Ltd. Our experiment started when the
animals were 2.0 to 3.5 months old. The animals were
then randomly assigned to two different high-energy diets
for 4.5 weeks:
1. Control HE (high energy) diet: 938.5 g/Kg version of
2018SC+F Harlan Tekled Ltd, USA, supplemented with
soybean oil to a final fat concentration of 6%.
2. PS-HOSO diet (custom-made, Harlan Tekled Ltd,
USA): 939 g/Kg version of 2018SC+F, Harlan Tekled Ltd,
USA, supplemented with PS-HOSO to a final fat concen-
tration of 6%. PS-HOSO constituted 4% w/w of total diet
and comprised 20% plant sterols esterified with high oleic
sunflower oil, and 15% canola oil-based diacylglycerol
(DAG).
This diet was made from a slightly concentrated version of
2018CS+F with 2% fat source basal mix. Standard milling
procedure was used to incorporate treatment oil into
standard gerbil chow.
The animals were housed in polypropylene cages at a con-
stant temperature of 22 to 23°C in a humidity-controlled
animal facility with a 12-h light/dark cycle. Free access to
water and food was provided. The animals were moni-
tored for body weight and tail blood glucose concentra-
tions, from the beginning of the intervention and
throughout (every 2-5 days), for 4.5 weeks. At endpoint,
half of each group of gerbils was deprived of food over-
night (16 h), and then all animals were lightly anaesthe-
tized with ketamine (Ketalar; Parke-Davis & Co., United
Kingdom) and exsanguinated by cardiac puncture. Blood
samples were collected for biochemical analyses. The liver
and epididymal fat were harvested and weighted. All
experimental procedures performed in the study were
authorized by the Institutional Animal Care and Use
Committee.

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Biochemical measures
Blood glucose concentration was determined by the enzy-
matic glucose analyzer, Glucometer Elite (Bayer, Elkhart,
IN) on blood samples taken from the tail vein. Plasma
samples that were obtained from EDTA-treated blood
samples by separation at 12,000 × g for 15 min, were
stored until lipid analysis, at -80°C. Then, an aliquot was
analyzed for total cholesterol, TG and HDL-cholesterol
concentrations by colorimetric methods (Boehringer
Mannheim, Mannheim, Germany). Insulin levels in
plasma were assessed by radioimmunoassay using a
human primary antibody (Phadesph; Kabi Pharmacia
Diagnostics, Uppsala, Sweden) according to the manufac-
turer's instructions.
Lipid extraction from liver samples
The liver samples were weighed and homogenized with
saline at a ratio of 1:5 (w:v) in plastic tubes on ice. Lipids
were extracted from an aliquot of the liver homogenate
according to the procedure of Folch et al. [24]. The total
amount of fat was calculated according to Leikin-Frenkel
et al. [25].
Data Analysis
Data was expressed as means ± SD and statistical signifi-
cance was taken as P < 0.05. Data was analyzed by one-
way analysis of variance (ANOVA) model for continuous
variables to assess the differences among the experimental
groups. Fisher exact test was used for comparison of non-
parametric variables, namely, gerbil survival, and the
number of gerbils in phase C, D, or presenting normogly-
cemia. The assumption of normal distribution and homo-
geneity of variance was tested for all outcome measures.
Accordingly, the differences between groups were evalu-
ated by two-tailed Student's t-test or Wilcoxon's signed-
rank test. Spearman's rank-order correlation coefficient
was used to evaluate the associations between changes in
insulin and in blood glucose levels at the intervention
endpoint. The statistical analyses were carried out by
using SPSS statistical software (SSPS Inc, Chicago, IL) ver-
sion 13.0.
Results
Animals
By the end of the 4.5 weeks intervention, 3 gerbils from
the control group had normal blood glucose levels (below
140 mg/dl), 22 presented typical phase C profile, one ger-
bil presented typical phase D symptoms (triglyceride lev-
els above 500 mg/dl) and 4 gerbils had died from
unrelated causes. Similarly, within the PS-HOSO diet
group, 6 animals had normal blood glucose levels, 19
demonstrated phase C characteristics, 4 gerbils presented
typical phase D symptoms, and one gerbil died. There
were no statistically significant differences between the
two groups in the numbers of animal in phase C, phase D,
animal with normal glucose levels, or dead animals. The
baseline body weights and blood glucose levels of the ger-
bils are shown in Table 1. No significant differences
among the various groups were evident.
Table 1: Baseline characteristics of tested gerbils1.
All tested gerbilsGerbils in phase C
Control PS-HOSOControlPS-HOSO
Total Non-
fasting
Fasting TotalNon-
fasting
FastingTotalNon-
fasting
FastingTotal Non-
fasting
Fasting
n 30 16 143014 1622 101219811
Body
weight
(gr)
162.40 ±
22.7
156.94 ±
24.5
168.64 ±
19.5
163.93 ±
26.2
169.43 ±
32.4
159.13 ±
19.2
167.14 ±
20.2
163.6 ±
25.7
170.08 ±
14.6
159.21 ±
27.5
160.50 ±
35.9
158.27 ±
21.3
Glucose
(mg/dL)
73.97 ±
8.0
72.63 ±
6.9
75.50 ±
9.2
72.40 ±
10.1
76.93 ±
11.0
*68.44 ±
7.5
73.77 ±
7.2
72.30 ±
7.6
75.00 ±
7.0
72.05 ±
9.6
75.75 ±
11.5
69.36 ±
7.3
Psammomys obesus were weighted and tested for blood glucose levels at the beginning of the experiment. Following 4.5 weeks of experimental diet
feeding, half of each animal group were deprived of food for 16 h. Statistical significance between baseline values of control and PS-HOSO
treatments was calculated for the gerbils that started the feeding period (all gerbils, total), for the subgroup that was later assigned to fast (all
gerbils, fasting) and the subgroup that was not assigned to fast (all gerbils, non-fasting). Statistical significance was also calculated for gerbils that
were in phase C at endpoint (phase C, total), for phase C gerbils that were not assigned to fast (phase C, non-fasting) and for phase C gerbils that
were assigned to fast (phase C, fasting).
1Values are means ± S.D
* P < 0.05.
PS-HOSO: plant sterol esterified with high oleic sunflower oil.

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Body weight, liver weight and fat accumulation of phase C
gerbils
PS-HOSO effect on body weight, liver weight and fat accu-
mulation is shown in Table 2. Animals fed PS-HOSO and
assigned to the fasting mode reduced (P = 0.045) 6.7% in
their body weight as compared to the control diet fed ani-
mals. The relative amount of white adipose tissue, as
determined by the ratio (w/w) of epididymal fat to liver
decreased (P = 0.044) by 16% compared to the corre-
sponding control group. None of the above mentioned
effects were observed in the non-fasting group. Liver
weight at the end of the study was 11% smaller (P =
0.039) among non-fasting animals in the control group,
as compared to non-fasting animals in the PS-HOSO
group. This difference was abolished in the fasting groups.
Analysis of liver fat weight to liver weight ratio didn't
reveal any significant difference between treatment groups
among non-fasting animals, indicating that the liver
weight difference didn't result from liver fat accumula-
tion. Interestingly, among fasting animals, this ratio was
27% lower (P = 0.04) in the PS-HOSO group in compari-
son with that of the control group.
Biochemical measures of Phase C gerbils
Treatment with PS-HOSO significantly reduced the
plasma levels of TC and non-HDL cholesterol by 24% (P
= 0.001) and 34% (P = 0.002), respectively, compared to
the control diet (Figure 1). When the fasting and non-fast-
ing groups of phase C gerbils were analyzed separately,
total cholesterol was reduced (P = 0.002) by 28% in the
PS-HOSO treated non-fasting animals, and tended to
decrease (P = 0.16) by 20.5% in the fasting group, as com-
pared to the control-fed animals (data not shown). Non-
HDL cholesterol concentrations tended to decrease (P =
0.064) by 37% in the PS-HOSO treated non-fasting ani-
mals, and decreased (P = 0.049) by 31% in the fasting ani-
mals, as compared to the control groups (data not
shown).
No significant differences in blood glucose levels between
the PS-HOSO-fed animals and the control diet-fed ani-
mals were detected at the end of the experimental diets
intake (Table 3). The glucose levels among all of phase C
gerbils, prior to fasting assignment, were elevated (280-
340 mg/dl) as indicated by the three last glucose measure-
ments (data not shown). The plasma levels of insulin
dropped (P < 0.0001) by 39% at the endpoint of the
experiment in the phase C PS-HOSO group, compared to
the control (Table 3). Comparable findings were shown
when the fasting and non-fasting groups were analyzed
separately in the fasting (44%; P < 0.0001) and non-fast-
ing (33%; P = 0.014) groups.
In order to test the possibility that the insulin level reduc-
tion observed in the PS-HOSO gerbils was caused by β-cell
degranulation and deterioration toward phase D, we plot-
ted plasma insulin vs. blood glucose for each separate ani-
mal (Figure 2). There was no correlation between the
insulin levels and the glucose levels in PS-HOSO animals,
regardless of the feeding regime, indicating that the insu-
lin reduction is probably not caused by β-cell degranula-
tion. Another indication for the animals not being in
phase D is the fact that they were screened for blood TG
levels of below 500 mg/dl.
Table 2: PS-HOSO effect on body weight, liver weight and fat accumulation of phase C gerbils1.
Control PS-HOSO
TotalNon-fastingFasting TotalNon-fasting Fasting
Bodyweight at endpoint (gr)219.05 ± 16.2214.60 ± 15.8222.75 ± 16.2 211.58 ± 19.8 216.63 ± 23.0 * 207.91 ± 17.3
Liver weight (gr) 8.23 ± 0.778.36 ± 0.80 8.12 ± 0.778.68 ± 1.19 * 9.40 ± 1.178.15 ± 0.93
Liver fat weight to liver weight ratio 0.11 ± 0.03 0.10 ± 0.020.11 ± 0.04* 0.08 ± 0.01 0.08 ± 0.08* 0.08 ± 0.01
Epididymal fat weight (gr) 6.35 ± 0.946.25 ± 0.96 6.43 ± 0.955.88 ± 1.73 6.54 ± 2.30 * 5.41 ± 1.06
Epididymal fat to liver weight ratio0.78 ± 0.140.75 ± 0.12 0.80 ± 0.150.68 ± 0.180.69 ± 0.25* 0.67 ± 0.14
Psammomys obesus were fed with Control or PS-HOSO as described in the method section and were monitored for body weight. Following 4.5
weeks of experimental diet feeding, half of each animal group was deprived of food for 16 h and then all animals were weighted and exsanguinated.
The liver and epididymal fat were harvested and weighted. Liver fat of 15 PS-HOSO treated animals (9 and 6 from the fasting and non-fasting
groups, respectively) and 16 control treated animals (7 and 9 from the fasting and non-fasting groups, respectively) was extracted and weighted.
Statistical significance between endpoint values of control and PS-HOSO treatments were calculated for all of phase C gerbils (total), and for fasting
and non-fasting phase C gerbils.
1Values are means ± S.D
*P < 0.05.
PS-HOSO: plant sterol esterified with high oleic sunflower oil.

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The effect of PS-HOSO feeding on the insulin levels is
strikingly evident from Figure 2 as the insulin measure-
ment values of the PS-HOSO-fed animals cluster in the
bottom end of the scale, compared to those of the control
diet-fed animals.
Discussion
The aim of this study was to test the effect of PS-HOSO, a
novel preparation of plant sterols esterified to high oleic
sunflower oil fatty acids mixed with 1,3-DAG, in a matrix
of high oleic sunflower oil, on the development of meta-
bolic syndrome and diabetic symptoms in Psammomys
obesus. PS have been intensely studied in recent years and
have shown to lower TC and LDL-C. These effects are
obtained mainly by reducing the intestinal absorption of
dietary and biliary cholesterol [26,9]. Current hypotheses
ascribe a vital role to the dysfunction of lipid metabolism
in the development of metabolic syndrome and type 2
diabetes [27,28]. Thus, we chose to test the effect of PS-
HOSO preparation in the gerbil Psammomys obesus which
serves as a model of nutritionally induced type 2 diabetes.
The progression of metabolic syndrome, and the subse-
quent diabetes mellitus, in these animals, resembles in
many aspects the development of metabolic syndrome
and diabetes in susceptible humans [19,20].
In this study, PS-HOSO treatment significantly reduced
the TC and non-HDL cholesterol among phase C gerbils,
compared to control diet treatment. These results are in
agreement with an animal study performed on Apolipo-
protein E-deficient mice [22] which demonstrated that
administration of PS esters of canola oil fatty acids in a
canola oil matrix strongly tended to lower TC levels. Sim-
ilarly, a human study performed on hypercholestero-
lemic, mildly overweight individuals [23] showed that
administration of PS esterified to olive oil in an olive oil
based matrix, significantly reduced LDL levels. These
results indicate that PS maintains their cholesterol lower-
ing properties when esterified to high oleic sunflower oil
fatty acids.
Interestingly, treatment with PS-HOSO significantly
reduced body weight and the amount of epididymal fat
among fasting phase C gerbils in comparison with the
control group. The reason for this reduction is not clear,
however, 1,3-DAG, a component of PS-HOSO, has been
previously reported to reduce the extent of postprandial
TG increase [29] and to reduce body fat in obese individ-
uals [30]. It is assumed that 1,3-DAG-rich diet is less read-
ily reesterified into chylomicron-TG moiety, thereby
Effect of PS-HOSO on plasma lipid levels of phase C gerbils
Figure 1
Effect of PS-HOSO on plasma lipid levels of phase C
gerbils. Psammomys obesus were fed with control (closed
bars) or PS-HOSO (open bars) as described in the method
section. Following 4.5 weeks of experimental diet feeding,
total cholestrol (A) and non-HDL cholesterol (B) were
measured. Results are means ± SD. Statistical significance
between endpoint values of control and PS-HOSO treat-
ments are * P-Value < 0.01, ** P-Value < 0.001. PS-HOSO:
plant sterol esterified with high oleic sunflower oil.
0
20
40
60
80
100
120
140
160
Total cholesterol (mg/dL)
Control
A
**
0
10
20
30
40
50
60
Non-HDL-Cholesterol (mg/dL)
B
*
PS-HOSO PS-HOSOControl
Table 3: PS-HOSO effect on glucose and insulin levels of phase C gerbils1.
Control PS-HOSO
Total Non-fastingFastingTotal Non-fastingFasting
Glucose at endpoint (mg/dL)169.41 ± 106.7271.70 ± 62.284.17 ± 33.4 180.16 ± 136.6 296.38 ± 143.095.64 ± 24.6
Insulin at endpoint (μIu/L)211.36 ± 61.0228.60 ± 67.1197.00 ± 54.0*** 128.37 ± 45.7* 153.63 ± 40.3 *** 110.00 ± 41.6
Psammomys obesus were fed with control or PS-HOSO as described in the method section and were monitored for blood glucose levels. Following
4.5 weeks of experimental diet feeding, half of each animal group was deprived of food for 16 h and then all animals were tested for blood glucose
and insulin levels. Statistical significance between endpoint values of control and PS-HOSO treatments were calculated for all of phase C gerbils
(total), and for fasting and non-fasting phase C gerbils.
1Values are means ± S.D
* P < 0.05., ** P < 0.0001.
PS-HOSO: plant sterol esterified with high oleic sunflower oil.