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First Department of Internal Medicine, Gifu University School of Medicine, Gifu 500, Japan; * Central Research Laboratories, Ajinomoto Co., Inc., Yokohama 244, Japan; and Laboratory of Nutritional Physiology, School of Food and Nutritional Sciences, The University of Shizuoka, Shizuoka 422, Japan
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
ACKNOWLEDGMENT
FOOTNOTES
LITERATURE CITED
ABSTRACT 
We examined the effect of dietary oils with different fatty acid compositions on the growth of visceral adipose tissue in rats. Rats were fed for 4 mo starting at weaning a basal diet containing (12 g/100 g diet) perilla oil rich in (n-3) polyunsaturated fatty acids (PUFA), safflower oil rich in (n-6) PUFA, olive oil rich in monounsaturated fatty acid, or beef tallow rich in saturated fatty acids. The amount of food consumed and body weight gain did not differ among the four dietary groups. The weight of the epididymal fat pad and the serum triglyceride concentration in perilla oil-fed rats were significantly lower (P < 0.05) than those of olive oil- and beef tallow-fed groups. The product of [(volume of individual adipocytes) × (number of adipocytes in epididymal fat pad)], which presumably represents total adipocyte volume in the fat pad, was significantly lower (P < 0.05) in perilla oil-fed rats than in beef tallow- and olive oil-fed groups. Expression of the late genes of adipocyte differentiation, peroxisome proliferator-activated receptor 
, adipocyte P2 and adipsin, was significantly (P < 0.05) down-regulated in epididymal fat tissue of rats that had been fed perilla oil rather than beef tallow or olive oil, whereas expression of the early gene, lipoprotein lipase, was not significantly affected. Greater levels (P < 0.05) of (n-3) PUFA in the membrane phospholipid fraction of the fat tissue were observed in perilla oil-fed rats than in the other dietary groups. These results suggest that perilla oil or (n-3) PUFA prevents excessive growth of adipose tissue in rats at least in part by suppressing the late phase of adipocyte differentiation.
INTRODUCTION
Obesity is associated with adipose hypertrophy and, in a more severe form, adipose hyperplasia (Faust et al. 1978
, Klyde and Hirsch 1979). A high fat diet induces both hypertrophy and hyperplasia of adipose tissue in rats. In other words, a high fat diet not only accelerates the filling process of pre-existing preadipocytes but stimulates the proliferation of adipose precursor cells (Klyde and Hirsch 1979). Recent advances have shown that adipocyte differentiation, from adipoblasts to adipocytes, is the key phenomenon underlying adipose hypertrophy (Butterwith 1994). The differentiation process consists of at least two phases (Ailhaud et al. 1992). The first (early) phase is the commitment of adipoblasts to preadipocytes, and this phase is associated with the induction of early genes, such as lipoprotein lipase (LPL).4 The second (late) phase is the differentiation of preadipocytes into mature adipocytes, and this phase is associated with the enhanced expression of adipocyte-specific genes, such as adipsin, adipocyte P2 (aP2), and glycerophosphate dehydrogenase (GPDH). Very recently, it has been suggested that subtypes of peroxysome proliferator-activated receptor (PPAR), an orphan nuclear receptor, are involved in adipocyte differentiation (Chawla and Lazar 1994, Chawla et al. 1994); PPAR and 
are induced in the late and early phases of the differentiation process, respectively (Chawla and Lazar 1994, Chawla et al. 1994).
Fatty acids have been shown to act as signal transducing molecules in the differentiation of adipocytes (Amri et al. 1994). Fatty acids enhance the expression of these late but not early genes and stimulate the postconfluent proliferation of preadipocytes in cultures (Amri et al. 1994). Therefore, fatty acids seem to regulate the late phase of adipocyte differentiation. Information is also available regarding the in vivo effect of fatty acids on adipose growth. In recent studies, attention has been focused on differences among the effects of various oils rich in several fatty acids, namely saturated fatty acids (SFA), monounsaturated fatty acids (MUFA), (n-6) polyunsaturated fatty acids (PUFA), and (n-3) PUFA. Previous studies suggested that diets rich in PUFA, especially those rich in (n-3) PUFA, prevent excessive adipose growth (Hainault et al. 1993, Shimomura et al. 1990), although the mechanism(s) of these effects are not fully understood.
In the present study, we examined the effect of different oils on the adipocyte differentiation-related genes. We examined the effect of perilla oil rich in an (n-3) PUFA, -linolenic acid, safflower oil rich in a (n-6) PUFA, linoleic acid, olive oil rich in a MUFA, oleic acid, and beef tallow rich in SFA, palmitic and stearic acids, on rats fed one of these oils after weaning.
MATERIALS AND METHODS
-linolenic acid). The mass ratios of SFA:MUFA:(n-6) PUFA:(n-3) PUFA in each oil was as follows: 56:43:1:0 in beef tallow, 12:81:6:1 in olive oil, 8:11:81:0 in safflower oil and 8:18:16:58 in perilla oil.
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Table 1. Fatty acid composition of dietary fats1,2 |
-tocopherol (0.25 g) was supplemented as an antioxidant to 1 kg of each diet. The food was sealed in air-tight plastic bags under nitrogen gas and stored at 
20°C until use. The food in the animal cages was shaded from light and changed every other day.
Experimental design.
Rats were divided into four groups of six rats each. The rats in each group were fed beef tallow diet, olive oil diet, safflower oil diet or perilla oil diet, and daily food intake was recorded. Rats were weighed weekly. All animals were killed after 12 wk of dietary treatment following 12 h of food depriviation. Rats were anesthetized with a mixture of ketamine and xylazine prior to killing and tissue dissection. Sera were collected from the inferior caval vein and immediately subjected to triglyceride and cholesterol analyses. Epididymal and perirenal adipose tissues of rats in each group were weighed and used for histologic examinations and analyses of fatty acid compositions and Northern blots. Care was taken to remove connective and other non-adipose tissue from the dissected adipose tissue prior to use. The study protocol was approved by the animal care committee of the Gifu University.
Adipocyte cell number.
Adipocytes were isolated from a portion of epididymal fat pads by collagenase digestion as described previously (Okuno et al. 1995, Tsutsumi et al. 1992). Isolated adipocytes were fixed with osmic acid and counted in a Coulter counter (Coulter Electronics, Hialeah, FL) (Cushman and Salans 1978). Total lipid per aliquot of adipose tissue was determined by the method of Dole (1956). Lipid content per cell was obtained by dividing total lipid per aliquot by the number of cells in an aliquot of the same size. Adipocyte number per fat depot was calculated by dividing the milligrams of lipid per fat depot by the micrograms of lipid per cell (Shepherd et al. 1993).
Fatty acid composition.
Fatty acid compositions of the phospholipid fraction of the adipose tissue were determined as described previously (Onogi et al. 1996). The fatty acids in the tissue were extracted by the method of Folch et al. (1951), and phospholipids were isolated according to the method of Rouser et al. (1967). Pentadecanoic acid (free acid) was added to the phospholipid fraction as an internal standard. The samples were subjected to methanolysis in 1.37 mol/L HCl in methanol at 80°C for 2 h under nitrogen. Fatty acid methyl esters were extracted with n-hexane and analyzed by gas chromatography (Shimadzu GC-17A, Shimadzu, Kyoto, Japan) with an HR-SS-10 column (Shimadzu). The oven temperature was programmed to increase from 50°C to 150°C at 7°C/min and from 150°C to 220°C at 3°C/min and to hold for a final 15 min. The identification and quantification of each fatty acid was made with authentic standard mixtures (Sigma Chemical, St. Louis, MO) using a CR-7A Chromatopac integrator (Shimadzu).
Northern blot analysis.
Total RNA was extracted according to the method of Chomczynski and Sacchi (1987). Northern hybridization was performed as described previously (Okuno et al. 1995), utilizing a 32P-labeled probe specific for LPL (Reynolds et al. 1990), adipsin (Spiegelman et al. 1983), aP2 (Amri et al. 1986), PPAR (Suruga et al. 1995) or glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Clones of LPL, adipsin and aP2 were kindly provided by William S. Blaner, Columbia University, New York, NY.
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Table 2. Liver and epididymal and perirenal fat pad weights of beef tallow-, olive oil-, safflower oil- and perilla oil-fed rats1,2 |
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Table 3. Serum concentrations of triglycerides and total cholesterol of beef tallow-, olive oil-, safflower oil- and perilla oil-fed rats1,2 |
) or the Welch t test (Armitage 1971) when variances were heterogeneous.
RESULTS AND DISCUSSION
, Su and Jones 1993). However, our results suggest that the difference in the absorption of the oils was not great enough to affect organ weight or growth. Thus, the suppressing effect of perilla oil on adipose growth was unlikely to be related to energy differences. One possibility is that there might be a compensatory increase in protein or water content of perilla oil-fed rats.
, Shimomura et al. 1990, Weintraub et al. 1988). This effect may not be specific for (n-3) PUFA, because both (n-6) and (n-3) PUFA have been reported to reduce similarly the serum triglyceride concentration (Weintraub et al. 1988). Diets rich in PUFA have been shown to increase lipoprotein lipase activities in heart and skeletal muscle, a key enzyme in removing of triglycerides from the blood stream (Engelberg 1966), resulting in lipid oxidization in the tissues (Shimomura et al. 1990); PUFA also facilitate the interaction of lipoprotein triglyceride with LPL by increasing the solubility of lipids in the circulating lipoproteins (Engleburg 1966, Weintraub et al. 1988). Therefore perilla oil may have suppressed serum triglyceride concentrations by inducing LPL activities in the present study.
Table 4.
Size and number of adipocytes in epididymal fat pad of beef tallow-, olive oil-, safflower oil- and perilla oil-fed rats1,2
Table 5.
Fatty acid composition of the phospholipid fractions of epididymal fat pad of beef tallow-, olive oil-, safflower oil- and perilla oil-fed rats1,2,3
ACKNOWLEDGMENT , late genes of the differentiation, was found in the perilla oil-fed rats compared with those fed beef tallow or olive oil. These results support the theory that perilla oil, or its unique component, (n-3) PUFA, suppressed principally the late phase of adipocyte differentiation.
Fig. 1.
Gene expression of adipocyte-related genes in epididymal fat pad of beef tallow-, olive oil-, safflower oil- and perilla oil-fed rats. A. Gene expression of lipoprotein lipase (LPL), adipsin, adipocyte P2 (aP2), peroxisome proliferator-activated receptor (PPAR) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in epididymal fat tissue of rats fed diet containing beef tallow, olive oil, safflower oil or perilla oil (12 g/100 g diet) for 4 mo. Total RNA was isolated from each fat tissue, fractionated through 1% agarose-formaldehyde gels, transferred to nylon membranes, and hybridized with 32P-labeled probes for either LPL (top bands), adipsin (second bands), aP2 (third bands), PPAR (fourth bands) or GAPDH (bottom bands). B. The relative intensity of each band was quantified by densitometric analysis and standardized with the band intensities of GAPDH. Values are means ± SD, n = 6. a,bvalues with different letters are significantly different (P < 0.05).
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-Linolenic acid is converted to EPA or DHA after enzymatic desaturation and elongation in the liver. The ratio of (n-3) PUFA to SFA, MUFA or (n-6) PUFA was significantly higher (P < 0.05) in the perilla oil-fed group than in the other groups (Table 5). Therefore, significant alterations in the fatty acid compositions were observed in the epididymal fat cell membrane phospholipids of rats fed perilla oil. Polyunsaturated fatty acid is preferentially incorporated into phospholipids in plasma membranes (Bremer and Norum 1982), and oral supplementation with (n-3) PUFA induces selective incorporation of (n-3) PUFA and competitively excludes other fatty acids in the membrane phospholipid fractions (Awad et al. 1990). These differences in the membrane compositions not only confirm that the respective fatty acids are absorbed and incorporated into the adipose tissue but also suggest several possible mechanisms underlying the effect of fatty acids on adipocyte differentiation.
) and that PUFA suppress fatty acid synthesis whereas SFA do not (Herzberg 1983). One possible mechanism underlying the effect of PUFA is the regulation of adipocyte differentiation. Recent works by Amri et al. (1991a, 1991b and 1994) showed that fatty acids of endogenous or exogenous origin play a central role in the regulation of adipocyte-related genes (Amri et al. 1991a, 1991b and 1994). Fatty acids do not affect the early stage of differentiation (the commitment of adipoblasts to preadipocytes); however, they trigger the late phase (the differentiation of preadipocytes to adipocytes) in adipocyte cultures (Amri et al. 1994). Our results support their observations in an animal model, because dietary supplementation with fatty acids affected the expression of the late genes, adipsin, aP2 and PPAR, rather than the early gene, LPL. Amri et al. (1994) also suggested that such an effect of fatty acids does not require their metabolism. In other words, fatty acids may exert their effect without being converted to metabolites or substrates for lipid synthesis. The regulation of adipocyte-related genes by PUFA was hypothesized to be mediated by an interaction of some fatty acid-binding proteins (or some transcription factors) with a "fatty acid response element" (Montalto and Bensadoun 1993); PPAR seems to be a likely candidate for such a transcription factor (Chawla and Lazer 1994, Safonova et al. 1994). Peroxisome proliferator-activated receptor is a member of the nuclear receptor superfamily related to thyroid and steroid receptors (Carson-Jurica et al. 1990, Evans 1988). Peroxisome proliferators, including drugs used clinically for hyperlipidemia (Gibson 1993), regulate gene transcription via activating PPAR (Evans 1988). PPAR, a subtype of the nuclear receptor, has been shown to be an adipose-specific gene and to be induced in the early adipocyte differentiation (Chawla et al. 1994). Thus, PPAR may be an early trigger of differentiation. PPAR, another subtype, is expressed in the late phase and stimulates terminal adipose differentiation (Chawla and Lazer 1994); PPAR also regulates lipid metabolism by forming a complex with another member of the nuclear receptor superfamily, retinoid X receptor (RXR), which binds to retinoic acid (Keller et al. 1993). The PPAR-RXR complex has been suggested to stimulate synergistically adipocyte differentiation (Safonova et al. 1994). Long-chain fatty acids regulate gene expression via PPAR and may be the natural ligands of PPAR (Göttlicher et al. 1992, Issemann et al. 1993). Therefore dietary fatty acids may have regulated adipocyte differentiation through PPAR in the present study. In fact, the expression of PPAR was affected by dietary fatty acids, as shown in Figure 1. Another possible mechanism involves the alteration of prostaglandin (PG) synthesis. Prostaglandins, especially PGI2 and PGF2
, are closely associated with the terminal differentiation of adipocytes (Gaillard et al. 1989, Nêgrel et al. 1989, Richelsen 1992). The (n-3) PUFA are strong cyclooxygenase inhibitors and thereby inhibit prostaglandin synthesis (Corey et al. 1983). In fact, we have shown that perilla oil suppressed a type 2 prostaglandin, PGE2 , in rats (Onogi et al. 1996). Therefore, in the present study, perilla oil may have suppressed adipocyte differentiation by down-regulating prostaglandin synthesis. Further study is necessary to test the possible role of prostaglandins in adipocyte differentiation.
). We have demonstrated here that perilla oil rich in a vegetable (n-3) PUFA (-linolenic acid) also has a beneficial effect on the prevention of adipose hypertrophy in male rats. The effect of perilla oil in female rats is now under investigation in our laboratory. -Linolenic acid is much more chemically stable than EPA or DHA (Cho et al. 1987). Therefore, daily consumption of perilla oil by humans seems advantageous.
, Onogi et al. 1996). We are now preparing for the clinical trial of an interventional study to prevent colon cancer with the use of perilla oil. We are also planning to examine the suppressive effect of perilla oil on the development of visceral adipose tissue in patients.
FOOTNOTES
-tocopheryl acetate, 60 mg menadione, 590 mg thiamine HCl, 590 mg riboflavin, 290 mg pyridoxine HCl, 2 mg cyanocobalamin, 5.88 g ascorbic acid, 10 mg d-biotin, 20 mg folic acid, 2.35 g d-calcium pantothenate, 2.94 g nicotinamide, 11.76 g inositol and 962.6 g lactose.
Manuscript received 19 November 1996. Initial reviews completed 8 January 1997. Revision accepted 13 May 1997.
LITERATURE CITED
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