Prevention of diet-induced obesity by safflower oil: insights at the levels of PPARα, Orexin, and Ghrelin gene expression of adipocytes in mice

Abstract

The aim of this study was to investigate the prevention of diet-induced obesity by a high safflower oil diet and adipocytic gene expression in mice. Forty 3-week-old C57BL/6 mice were randomly divided into three groups: control group (CON, 5% lard + 5% safflower oil), high lard group (LAR, 45% lard + 5% safflower oil), and high safflower oil group (SAF, 45% safflower oil + 5% lard). After 10 weeks, 10 mice of the LAR group were switched to high safflower oil diet (LAR–SAF). Ten weeks later, glucose tolerance tests were performed by intraperitoneal injection of glucose. Circulating levels of lipid and insulin were measured and white adipose tissues were taken for gene chip and reverse transcriptase–polymerase chain reaction analysis. The LAR group showed higher body weight, adiposity index, insulin, and lipids than the CON group (P < 0.05). The body weight in the LAR–SAF group decreased after dietary reversal. The plasma biochemical profiles decreased in the LAR–SAF and SAF groups (P < 0.05) compared with those of the LAR group. The blood glucose level of the LAR–SAF group was reduced during intraperitoneal glucose tolerance test compared with that of the LAR group. The LAR–SAF group had lower levels of Orexin and Ghrelin gene expression, whereas the level of PPARα gene expression was significantly enhanced compared with that of the LAR group. So, the SAF diet can alter adipocytic adiposity-related gene expression and result in effective amelioration of diet-induced obesity.

safflower oil, adiposity, PPARα, Orexin, Ghrelin

Introduction

Obesity is a metabolic disease that develops via the cooperation of genetic predisposition and lifestyle-related factors, including unscientific diet customary and physical inactivity [1]. Adipose tissue, the energy reserve organ, plays an important role in regulating energy metabolism in organisms [2]. Mature white adipocytes express proteins that regulate lipid and carbohydrate metabolisms, secretary cytokines, and hormones that affect energy metabolism in other tissues. Adipocyte dysfunction is strongly associated with the development of obesity. It is accepted that specific regulation of gene expression in adipocytes is one of the most important targets for the intervention of obesity. Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors that control lipid oxidation, adipocyte differentiation, glucose and lipid storage, and inflammation [3,4]. Ghrelin is an endocrine link connecting physiological processes regulating nutrition, body composition, growth, and energy balance [5,6]. Exogenous Ghrelin stimulates food intake and promotes energy storage [7]. Orexin is a peptide involved in the regulation of feeding and sleep. Central infusion of Orexin accelerates feeding behavior [8]. There are few reports about the relationship of PPARα, Ghrelin, and Orexin gene expression in adipocyte with obesity.

Many animal and clinical studies have shown that the dietary fat content is a major determinant of body adiposity [9,10]. Fat has traditionally been regarded to be important as a calorie-dense nutrient and as a source for essential fatty acids. The dietary fat type, rather than just the percentage of fat calories, is a critical factor in relation to diet-induced obesity [11]. Dietary fatty acids can be classified into four types: saturated and monounsaturated fatty acids, and n-6 and n-3 polyunsaturated fatty acids (PUFAs). The n-3 and n-6 PUFAs are regarded as essential fatty acids. Polyunsaturated fats can modify the responsiveness of isolated white adipocytes to insulin [12]. Compared with saturated fatty acids, dietary fats rich in n-3 [13] PUFAs have been reported to cause less body fat accumulation in rats. An n-3 PUFA-rich diet is less adipogenic [14] because of coordinated induction of fatty acid oxidation genes through PPAR [15] and suppression of lipogenic genes through sterol-responsive element-binding proteins (SREBP) [16]. A high oleic acid-rich safflower oil (rich in monounsaturated fatty acids) diet was shown to be as effective as high n-3 PUFA diets in lowering body fat accumulation in meal-fed Sprague–Dawley rats [17]. However, relatively few studies have focused on the effect of dietary n-6 PUFAs on the gene expression of white adipose tissues and it is even less well known whether established obesity, as a result of a high intake of dietary saturated fatty acids, can be ameliorated.

Safflower is a very ancient crop and is used as a type of herb in Chinese medicine. Safflower oil contains typical n-6 PUFAs. The main constituent, linoleic acid, accounts for 74% of the fatty acid content. Compared with other fat types, safflower oil has advantages, such as better stability during cooking, adequate essential fatty acid content [about 75% is 18:2 (n-6)], very low saturated fatty acid content, and a lower price than olive oil.

In the present study, we compared safflower oil with a source of saturated fatty acids (lard) and investigated the prevention of diet-induced obesity by safflower oil and also explored the gene expression of adipocytes in C57BL/6 mice using gene-chip microarray and reverse transcriptase–polymerase chain reaction (RT–PCR) analysis. It was found that the safflower oil diet ameliorated diet-induced obesity of diet-induced obese mice and altered adipocytic PPARα, Ghrelin, and Orexin gene expression and was less adipogenic than a diet high in saturated fat.

Materials and Methods

Animals

Forty 3-week-old C57BL/6 male mice were obtained from the Vital River Laboratory Animal Technology Company (Beijing, China). The mice were housed in a temperature-controlled room (20 ± 2°C) with a 12-h light–dark cycle (lights on at 07:00 a.m.) and were given ad libitum access to food and water throughout the study. The study was carried out in accordance with the Guide for the Care and Use of Laboratory Animals of Harbin Medical University.

Diets and experimental procedure

The mice were fed standard laboratory chow for the first week to allow them to adjust to the new environment. Then they were randomly divided into three groups: two groups of mice were fed diets enriched with either high lard (LAR group, 20 mice, 45% lard + 5% safflower oil) or high safflower oil (SAF group, 10 mice, 45% safflower oil + 5% lard in diet) (Shanghai Young sun Foods Limited Company, Shanghai, China) and a group of mice were fed a low fat diet (CON group, 10 mice, 5% lard + 5% safflower oil). The dietary composition of each diet in terms of calorie content is shown in Table 1. The diets were freshly made every week and stored at 4°C. The mice were housed individually and their weights were measured weekly. After 10 weeks on their respective diets, 10 mice in the LAR group were switched to the SAF diet (LAR–SAF group) and all the mice were fed their respective diets for a further 10 weeks (Week 20). Then glucose tolerance tests were performed by intraperitoneal injection of glucose (2 g/kg body weight) in mice and blood glucose was measured using an Accu-Chek Performa System (Roche, Germany). The mice were sacrificed and blood was collected from the orbital sinus. Plasma was separated by centrifugation and frozen at −20°C. Plasma triglyceride (TG), total cholesterol (T-ch), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) were determined using respective assay kits. Insulin was determined by radioimmunoassay (Linco Research Inc., St. Louis, USA).

Table 1

Major dietary composition by calories (%)

Group	CON (10 mice)	LAR (20 mice)	SAF (10 mice)
Carbohydrate	69	30	30
Corn starch	25	16.3	16.3
Sucrose	35.5	8	8
Maltodextrin	8	5.5	5.5
Protein	21	20	20
Casein	20.7	19.8	19.8
Fat	10	50	50
Larda	5	45	5
Safflower oilb	5	5	45
Total energy (kcal/kg)	4000	4000	4000

Numbers in bold font represent the total proportion of protein, carbohydrate, and fat. The other numbers indicate the individual sources of protein, carbohydrate, and fat by calories in percent. ^aFatty acid composition of lard, as analyzed by GC: 16:0, 24.5%; 16:1, 3.1%; 18:0, 14.9%; 18:1, 42.7%; 18:2, 9.8%; and others, 5.0%. ^bFatty acid composition of safflower oil, as analyzed by GC: 16:0, 7.7%; 16:1, 0%; 18:0, 2.5%; 18:1, 14.7%; 18:2, 74.2%; and others, 0.9%.

RNA isolation, cRNA synthesis, and labeling

At Week 20, white adipose tissues (epididymal, perirenal, and retroperitoneal) were dissected, weighed, and snap-frozen in liquid nitrogen immediately. The adiposity index for individual animals was calculated by dividing the sum of the weight of the dissected fat depots by the body weight (minus the weight of the fat depots). Six white adipose tissues were also taken from three random mice in the LAR group and three random mice in the LAR–SAF group. RNA was isolated from each adipose tissue sample using TRIzol reagent (Invitrogen, USA). RNA quality was assessed by denaturing agarose gel electrophoresis. cRNA was labeled and purified using the methods of Schena et al. [18] and Czechowski et al. [19] with the Array grade cRNA cleanup kit (Super Array Bioscience Corporation, USA).

Hybridization and data analysis

cRNA was hybridized to an Oligo GEArray gene chip (Super Array Bioscience Corporation) according to the manufacturer's protocol. The gene chip was detected with the Chemiluminescent detection kit (Super Array Bioscience Corporation). We used the web-based, completely integrated, GEArray expression analysis suite to perform the data analysis in accordance with the manufacturer's instructions. The operations were performed six times.

Reverse transcriptase–polymerase chain reaction

Total cellular RNA of white fatty tissues of the CON, LAR, and LAR–SAF groups was prepared using the TRIzol reagent (Invitrogen). Two micrograms of total RNA were reverse transcribed using Moloney murine leukemia virus reverse transcriptase and an antisense primer to generate cDNA under standard conditions. cDNA samples were amplified by PCR in an MJ Research Thermocycler (Waltham, USA). The respective forward and reverse primers used were as follows: PPARα, 5′-CGGGTAACC-TCGAAGTCTGA-3′ and 5′-CTAACCTTGGGCCACACC-T-3′; Orexin, 5′-TCCTGCCGTCTCTACGAACT-3′ and 5′-GGGATGTGGCTCTAGCTCTG-3′; Ghrelin, 5′-TTGA-GCCCAGAGCACCAGAAA-3′ and 5′-AGTTGCAGAGG-AGGCAGAAGC-3′. The reaction consisted of 30 cycles of denaturation for 1 min at 94°C, annealing for 1 min at 58°C, and elongation for 1 min at 72°C. The PCR products were analyzed by electrophoresis on a 1% agarose gel. Quantitative densitometric analysis was performed using a computerized image analysis system (Bio-Rad, USA). All the measured PCR products were normalized to the amount of cDNA of β-actin in each sample.

Statistics analysis

The results were analyzed using unpaired t-test. Values are presented as the mean ± SD. Statistical significance was defined as P < 0.05. Oligo GEArray gene chip studies considered the genes that showed changes in expression exceeding 2 folds (up- or down-regulation), as recommended by the manufacturer.

Results

Effects of switching from the high lard diet to the high safflower oil diet for 10 weeks

Table 2 shows the body weight at the start of the study, at Week 10 and at Week 20, TG, T-ch, HDL-C, LDL-C, and insulin concentrations, and the adiposity index for each group at Week 20. At the beginning of the study, there were no differences in body weight between groups. At Week 20, the LAR group showed significant differences compared with the CON group (P < 0.05). Of note, indices for the LAR–SAF and SAF groups showed statistically significant decreases compared with those in the LAR group and were incomparable with those in the CON group. The body weight in the LAR–SAF group at Week 20 was significantly lower (P < 0.05) than that at Week 10, i.e. the time point at which the diet was switched. This indicates that the mice in the LAR–SAF group lost weight after switching from the LAR diet to the SAF diet.

Table 2

Effects of dietary fat on body weight and plasma biochemical profiles at Week 20

Group	CON	LAR	LAR–SAF	SAF
Body weight at start (g)	17.15 ± 1.81	17.67 ± 1.97	18.10 ± 1.99	19.23 ± 2.08
Body weight at Week 10 (g)	25.38 ± 2.65	31.27 ± 3.31*	32.09 ± 3.42*	25.93 ± 2.69
Body weight at Week 20 (g)	27.17 ± 2.72	36.53 ± 3.81*	28.54 ± 2.95**	27.53 ± 3.17**
TG (mM)	0.67 ± 0.13	0.83 ± 0.18*	0.62 ± 0.11**	0.64 ± 0.14**
LDL-C (mM)	1.59 ± 0.20	2.47 ± 0.28*	2.02 ± 0.33**	1.89 ± 0.52**
T-ch (mM)	2.22 ± 0.29	3.51 ± 0.40*	2.69 ± 0.48**	2.76 ± 0.73**
HDL-C (mM)	2.02 ± 0.25	2.65 ± 0.22	2.45 ± 0.38	2.38 ± 0.64
Insulin (ng/ml)	0.45 ± 0.10	0.82 ± 0.26*	0.33 ± 0.17**	0.50 ± 0.21**
Adiposity index	2.81 ± 0.63	6.97 ± 2.30*	3.78 ± 0.87**	3.29 ± 0.87**

Data are presented as the mean ± SD (n = 10). The results were compared using unpaired t-test. *P < 0.05 significantly different from the CON group. **P < 0.05 significantly different from the LAR group.

Intraperitoneal glucose tolerance test

The effects of safflower oil on blood glucose levels were measured during intraperitoneal glucose tolerance test in the CON, LAR, and LAR–SAF groups (Fig. 1). After 10 weeks of treatment with SAF diet, the LAR–SAF group exhibited significant reductions in blood glucose concentrations following intraperitoneal glucose administration compared with the obese LAR group. There was no significant difference between the CON and the LAR–SAF groups. These results indicated that safflower oil may improve glucose tolerance in mice with diet-induced obesity.

Figure 1

Changes in blood glucose levels during intraperitoneal glucose tolerance test Mice were intraperitoneally injected glucose (2 g/kg body weight). All values are expressed as the mean ± SD. *P < 0.05 compared with the LAR group.

Gene chip analysis

We synthesized the results of the six gene chips. Tables 3 and 4 show the up- and down-regulated genes in the adipocytes of the LAR–SAF group compared with the LAR group. Seventeen genes were up-regulated and six genes were down-regulated with more than 2-fold changes in expression. Switching from the LAR to the SAF diet induced the expression of cell signaling factor and receptor genes (GHSR, GHR, IL-1R, IL-1, HRH1, HRH3, MC3R, ADRB3, GLPR, NPYR, and OPRM) and peptide genes (Ghrelin, Orexin, GRP, UCN, NMB, and ADCYAP1). The down-regulated genes included those of hormones and receptors (GCG, SST, CNR, MOXR, NR3C, and PPARα).

Table 3

Adipose tissue genes showing decreased expression of the LAR–SAF group versus the LAR group

Accession no.	Fold change	Protein	Function
Orexigenic genes
NM_021488	0.17	GHRL	Cell growth/appetite
NM_177330	0.36	GHSR	Cell growth/receptor
NM_010410	0.29	HCRT	Appetite/metabolism
NM_008731	0.31	NPYR	Signal transduction/receptor
NM_011013	0.38	OPRM	Receptor
Anorectic genes
NM_010284	0.33	GHR	Cell growth/receptor
NM_021332	0.38	GLPR	Signal transduction/receptor
NM_175012	0.40	GRP	Signal transduction
NM_008285	0.26	HRH1	Signal transduction/receptor
NM_133849	0.33	HRH3	Signal transduction/receptor
NM_010554	0.32	IL-1	Signal transduction
NM_008362	0.43	IL-1R	Signal transduction/receptor
NM_008561	0.40	MC3R	Signal transduction/receptor
NM_026523	0.39	NMB	Signal transduction
AF331517	0.21	UCN	Signal transduction
Genes involved in energy expenditure
NM_009625	0.47	ADCYAP1	Growth/metabolism
NM_013462	0.39	ADRB3	Signal transduction/receptor

GHRL, Ghrelin; GHSR, growth hormone secretagogue receptor; HCRT, hypocretin/Orexin; NPYR, neuropeptide Y receptor Y2; OPRM, opioid receptor, mu; GHR, growth hormone receptor; GLPR, glucagon-like peptide 1 receptor; GRP, gastrin releasing peptide; HRH1, histamine receptor H1; HRH3, histamine receptor H3; IL-1, interleukin 1 alpha; IL-1R, interleukin 1 receptor, type I; MC3R, melanocortin 3 receptor; NMB, neuromedin B; UCN, urocortin 2; ADCYAP1, adenylate cyclase activating polypeptide 1; ADRB3, adrenergic receptor, beta 3.

Table 4

Adipose tissue genes showing enhanced expression of the LAR–SAF group versus the LAR group

Accession no.	Fold change	Encoding protein	Function
Orexigenic genes
NM_009924	4.75	CNR	Signal transduction/receptor
NM_021325	2.22	MOXR	Signal transduction/receptor
NM_008173	3.93	NR3C	Signal transduction/receptor
Anorectic genes
NM_008100	5.00	GCG	Metabolism
NM_009215	3.59	SST	Growth/digestion
Genes involved in energy expenditure
NM_011144	30.86	PPARα	Growth/metabolism/receptor

CNR, cannabinoid receptor 2 (macrophage); MOXR, antigen identified by monoclonal antibody MRC OX-2 receptor; NR3C, nuclear receptor subfamily 3, group C, member 1; GCG, glucagon; SST, somatostatin; PPARα, peroxisome proliferator-activated receptor α.

Expression of adipose PPARα, Ghrelin, and Orexin mRNA

To confirm the microarray results, we performed mRNA measurements by using RT–PCR for PPARα, Ghrelin, and Orexin genes that showed a marked change in expression in adipose tissue. Figures 2 and 3 show that the LAR–SAF group had lower levels of Ghrelin and Orexin mRNA expression, meanwhile the PPARα mRNA expression was enhanced compared with that of the LAR group (P < 0.05). In the LAR group, Orexin and Ghrelin mRNA expression was increased and PPARα mRNA expression was reduced compared with that of the CON group (P < 0.05). And the LAR–SAF group had higher level of PPARα mRNA expression than that of the CON group (P < 0.05).

Figure 2

The mRNA expression levels of PPARα, Orexin, and Ghrelin genes in white adipose tissue of the CON, LAR, and LAR–SAF groups at Week 20 All values are expressed as the mean ± SD of relative density units (RDU) using β-actin as a reference. ^#P < 0.05 compared with the LAR group; *P < 0.05 compared with the CON group.

Figure 3

The mRNA expression levels of PPARα, Orexin, and Ghrelin genes in white adipose tissue of the CON, LAR, and LAR–SAF group at Week 20 Representative PCR bands are shown here.

Discussion

Adipocyte dysfunction plays an important role in the development of obesity and insulin resistance. Some drugs are used to treat obesity-related metabolic diseases. Safflower is an ancient crop and is used in Chinese herbal medicine. According to the ‘Compendium of Materia Medica’, safflower can ‘promote blood circulation, moisturize, induce analgesia, reduce swelling, and regulate menstruation’. Safflower oil is edible oil rich in n-6 PUFAs. The main constituent is linoleic acid, which accounts for 74% of the lipid content. Safflower oil can lower blood pressure, prevent hypertension and hyperlipidemia, and can aid lipid metabolism in vivo [20,21]. High safflower oil feeding significantly decreased 2-deoxyglucose uptake and glucose tolerance compared with high fat diet containing a mixture of soybean oil, hydrogenated coconut oil, and sunflower oil in rats [22]. In another study, safflower oil significantly reduced hepatic endogenous glucose production and glycogenolysis compared with palm oil in obese non-diabetic subjects [23]. Thus, we used DNA microarrays and RT–PCR to investigate the prevention of diet-induced obesity by safflower oil and gene expression of adipocytes in mice.

We found that the mice fed on the high lard diet (LAR group) showed marked obesity, hyperglycemia, and hyperinsulinemia with abnormal lipid profiles at Week 20 (Table 2). The mice fed on the safflower oil diet for 20 weeks (SAF group) showed neither obesity nor carbohydrate and lipid metabolic disorders. These findings agree with the results of Takeuchi et al. [17] that a diet comprising 20% (w/w) high oleic acid-rich safflower oil was less adipogenic in rats than a diet containing 20% lard. After switching the LAR–SAF group from the high lard to the high safflower oil diet, the mice showed significant weight loss. Accordingly, this reveals that the induction of obesity in mice with a high saturated fat diet for 10 weeks is reversible by switching to a diet comprising high levels of n-6 PUFAs. Moreover, the glucose tolerance, insulin, and lipid concentrations in the LAR–SAF group were significantly lower than those in the LAR group, which indicates that the carbohydrate and lipid metabolisms were improved in the LAR–SAF group. These differences between the LAR–SAF and the LAR groups suggest that safflower oil could be used in the prevention or treatment of obesity. This is different to the result of a study by Zhou et al. [24] who reported that long-term intake of a diet containing high levels of unsaturated fatty acids and sucrose could induce insulin resistance in aging rats. These differences may be due to the different experimental conditions. They fed the aging rats on a diet based on tallow and bean oil for 12 and 24 weeks. In our current study, there was no difference in the HDL-C level of each group, the reason for which is currently unknown.

Obesity is a chronic disease involving adverse changes in the expression of several genes [25,26]. In this experiment, we used GEArray gene chip and found that the expression of a number of genes related to metabolism had changed in the adipose tissue of the mice in the LAR–SAF group (Tables 3 and 4). These genes included orexigenic genes, anorectic genes, and some genes involved in energy expenditure. Importantly, the adipocytic PPARα gene expression was up-regulated and the Orexin and Ghrelin gene expression was down-regulated after safflower oil dietary feeding. The results of RT–PCR confirmed the microarray results (Figs. 2 and 3). Moreover, the LAR group had higher levels of Orexin and Ghrelin mRNA expression and lower level of PPARα mRNA expression than the CON group. PPARα, Orexin, and Ghrelin are important peptides regulating feed behavior or energy metabolism. Orexin and Ghrelin can stimulate feed behavior [5−8]. PPARα can enhance β-oxidation in white adipocytes [27]. Thus, it may be due to the change of the adipocytic PPARα, Orexin, and Ghrelin gene expression that safflower oil prevents diet-induced obesity of mice. However, it remains to be determined whether the effects of the SAF diet on adipocytic PPARα, Orexin, and Ghrelin gene expression are direct or indirect. These results are different to the study that the rats fed on 30% safflower oil and mice fed with 20% safflower oil showed significantly higher body weight, white adipose tissue weight, serum leptin, and hepatic PPARα mRNA expression [28]. But these results are consistent to another experiment that a high oleic acid-rich safflower oil diet resulted in less retroperitoneal white adipose tissue mass, increased mRNA expression of PPARα target genes in the liver, and lower mRNA expression of SREBP-1c in epididymal white adipose tissue, compared with a high butter fat diet [29]. Interestingly, the RT–PCR results showed that the LAR–SAF group had higher level of PPARα mRNA expression than the CON group. This suggests that safflower oil diet excessively induced adipocytic PPARα gene expression. The specific mechanism inducing these results needs to be investigated further.

Conclusion

The high lard diet induced metabolic disorders in mice. The SAF diet can potently alter adipocytic adiposity-related gene expression and result in effective amelioration of diet-induced obesity. Safflower oil diet may prevent obesity. The mechanisms involved need to be investigated in further studies.

Funding

This work was supported by the grants from the Special Fund of Doctor Station of University of Education Department of China (no. 200802260007), the Innovation Project of Main Instructor of Education Department of Heilongjiang Province, China (no. 1054G024), and the Important Project of Overseas Scholar of Education Department of Heilongjiang Province, China (no. 1152hq34).