Physiological Functions of Iso-Type Short-Chain Fatty Acid and Omega

Physiological Functions of iso-type Short-Chain Fatty Acid and Omega 3 Polyunsaturated Fatty Acids Containing Oil in Obese OLETF Rats Bungo Shirouchi 1, Koji Nagao 1, Kenta Furuya 1, Toshiharu Nagai 2, Kenji Ichioka 2, Shigeru Tokairin 2, Yasuhiro Iida 3 and Teruyoshi Yanagita 1＊ 1 Laboratory of Nutrition Biochemistry, Department of Applied Biochemistry and Food Science, Saga University (Saga 840-8502, JAPAN) 2 Tsukishima Foods Industry Co., Ltd. (Tokyo 134-8520, JAPAN) 3 Japan Institute for Oils & Fats & Foods Inspection (Tokyo 103-0007, JAPAN) Abstract: It has been known that tissues of porpoise contain unique structured-lipids as combination of iso- valeric acid (iso-C5:0) and omega 3 polyunsaturated fatty acids (ω3 PUFAs). It is well known that ω3 PU- FAs have lipid-lowering effects in animal and human studies. Although branched chain fatty acids have been interested in their unique functions, there is no data concerning the effect of iso-C5:0 on lipid metabolism. In this study we investigated the effect of structured-lipids from porpoise adipose tissue (porpoise oil) on lipid metabolism in Otsuka Long-Evans Tokushima Fatty (OLETF) rats. For 4 weeks, rats were fed semisynthetic diets containing either 10% corn oil or 5% corn oil plus 5% porpoise oil. After feeding period, the porpoise oil diet signiﬁ cantly alleviated hepatic triglyceride accumulation compared with the control diet in OLETF rats. Although serum triglyceride level increased, serum level of adiponectin that can protect liver function and alleviate abnormalities of lipid and glucose metabolism increased in rats fed porpoise oil diet. In conclusion, results from the present study suggest that porpoise oil feeding prevents the development of fatty liver disease through the enhancement of lipoprotein secretion and increase of adiponectin production in obese rats.

Key words: iso-type short-chain fatty acid, ω3 polyunsaturated fatty acids, adiponectin, OLETF rats

1 INTRODUCTION studied6-7）. Differential effects have arisen with respect to Lifestyle-related diseases, such as hyperlipidemia, ath- individual fatty acids8-10）. Short-chain fatty acids（SCFAs）, erosclerosis, diabetes mellitus, and hypertension, are wide- which generally consist of C2-C5, are produced as a result spread and increasingly prevalent diseases in industrialized of colonic bacterial fermentation of dietary fi ber. Dietary fi - countries and contribute to the increase in cardiovascular ber has several beneficial effects, including the improve- morbidity and mortality1, 2）. Accompanied by the rapid in- ment of glucose tolerance, lowering of plasma lipid levels, crease in the number of elderly people, it becomes impor- and the reduction in fat storage11）. Some studies have sug- tant not only medically but also socioeconomically. Al- gested that increased SCFAs production in bowel might be though the pathogenesis of lifestyle-related diseases is partly responsible for the beneficial effects of dietary fi- complicated and the precise mechanisms have not been ber11）. In fact, it has been reported that feeding of acetic elucidated, obesity has emerged as one of the major car- acid（C2:0）reduces hyperglycemia by activating AMP-acti- diovascular risk factors according to epidemiologic stud- vated protein kinese in the liver of diabetic mice12）and that ies3-5）. administration of propionic acid（C3:0）inhibits cholesterol Because diet, especially dietary fat, has been recognized biosynthesis and fatty acid biosynthesis in isolated rat he- as contributing to the development and prevention of obe- patocytes13）. In addition, branched chain fatty acids have sity, the infl uence of quantities and qualities of dietary fats been interested in their unique functions14-17）. For example, on the pathogenesis of obesity-related disorders has been valproic acid（VPA; 2-n-propylpentanoic acid）, an eight-

＊Correspondence to: Teruyoshi Yanagita, Laboratory of Nutrition Biochemistry, Department of Applied Biochemistry and Food Science, Saga University, Saga 840-8502, JAPAN E-mail: [email protected] Accepted January 12, 2010 (received for review December 3, 2009) Journal of Oleo Science ISSN 1345-8957 print / ISSN 1347-3352 online http://www.jstage.jst.go.jp/browse/jos/ 299 B. Shirouchi , K. Nagao, K. Furuya et al.

carbon branched-chain fatty acid, is an analogue of valeric Table 1 Fatty Acid Composition of Experimental acid（C5:0）and has been used for the treatment of epilepsy, Diets bipolar disorder and migraine prophylaxis15-17）. Recently, Fatty acid Control diet PO diet therapeutic roles of VPA have more been proposed in can- cer, Alzheimer's disease and HIV treatment. It is also well (wt %) known that omega 3 polyunsaturated fatty acids（ω3 PU- iso-C5:0 n.d. 10.0 FAs）, such as eicosapentaenoic acid（EPA; C20:5）and doc- iso-C14:0 n.d. 1.1 osahexaenoic acid（DHA; C22:6）, are abundant in fish, C14:0 n.d. 3.9 shellfish, and sea mammals, and evidence from animal models and human studies have suggested that ω3 PUFAs iso-C15:0 n.d. 3.3 have lipid-lowering effects18,19）. C15:0 n.d. 0.9 The concept of a“ structured-lipid” implies modifi cation iso-C16:0 n.d. 1.1 of the fatty acid composition and/or their location in the C16:0 11.1 8.4 glycerol backbone, and improvement of the physical and/or physiological properties of dietary lipids. For example, C16:1 0.1 5.9 feeding of a structured triglyceride, containing both medi- C18:0 2.0 1.0 um-chain fatty acids and long-chain fatty acids in the same C18:1 29.7 18.6 molecule, reduced body fat accumulation and increased C18:2 55.0 27.5 postprandial hepatic β-oxidation of fatty acids compared 20, 21 C18:3 3 0.9 0.5 with long-chain triglyceride in rats ）. Additionaly, feed- ω ing of seal oil, in which EPA and DHA are mainly located in C20:0 0.4 0.2 sn-1,3 positions of triglyceride, more effectively reduced C20:1 0.3 0.2 plasma and liver triglyceride levels through the suppres- C20:5 ω3 n.d. 1.1 sion of fatty acid synthesis than fi sh oil, in which EPA and C22:0 0.1 0.1 DHA are mainly located in sn-2 position, in rats22, 23）. It has been known that tissues of porpoise contain unique struc- C22:1 n.d. 0.7 tured-lipids as combination of iso-valeric acid（iso-C5:0）and C22:6 ω3 n.d. 1.8 24, 25） ω3 PUFAs . Therefore, in the present study, we investi- Others 0.4 13.7 gated the physiological functions of iso-C5:0 and ω3 PUFAs n.d.: not detected containing oil which was extracted from porpoise adipose tissue（porpoise oil）in Otsuka Long-Evans Tokushima Fatty （OLETF）rats. OLETF rats develop a syndrome with multi- synthetic diets were prepared according to recommenda- ple metabolic and hormonal disorders that shares many tions of the AIN-7628）and contained the following（in weight features with human obesity26, 27）. OLETF rats have hyper- %）: casein, 20; cornstarch, 15; cellulose, 5; mineral mixture phagia, because they lack receptors for cholecystokinin, （AIN-76）, 3.5; vitamin mixture（AIN-76）, 1; DL-methionine, and become obese and develop obesity-related lipid abnor- 0.3; choline bitartrate, 0.2; fat, 10; and sucrose, 45. Por- mality. poise oil was extracted from porpoise（Phocoenoides dal- li）adipose tissue. The fatty acid compositions of control diet and PO diet are shown in Table 1. The rats consumed each diet for 4 weeks. At the end of the feeding period, the 2 MATERIALS AND METHOD rats were sacrifi ced by aortic exsanguination under diethyl 2.1 Animals and diets ether anesthesia after 9 h starvation period. Epididymal, All aspects of the experiment were conducted according perirenal, omental, and waist subcutaneous white adipose to the guidelines provided by the ethical committee of ex- tissue（WATs）and the liver were excised immediately, and perimental animal care at Saga University. Male OLETF serum was separated from the blood. rats aged 4 weeks were provided by Tokushima Research Institute（Otsuka Pharmaceutical, Tokushima, Japan）. The 2.2 Measurement of serum parameters rats were housed individually in metal cages in a tempera- Serum levels of triglyceride, cholesterol and phospholip- ture-controlled room（24ºC）under a 12-h light/dark cycle. id were measured using commercial enzyme assay kits After a 1-week adaptation period, the rats were assigned to （Wako Pure Chemicals, Tokyo, Japan）. Serum levels of adi- two groups（six rats each）that were fed one of two diets: a ponectin and leptin were measured using commercial rat semisynthetic diet supplemented with 10% corn oil（Con- ELISA kits（Otsuka Pharmaceutical Co. Ltd., Tokyo, Japan; trol group）or a semisynthetic diet supplemented with 5% Yanaihara Institute Inc., Shizuoka, Japan, respectively）. corn oil plus 5% porpoise oil（PO group）. The basal semi- 300 J. oleo Sci. 59, (6) 299-305 (2010) Physiological Functions of Porpoise Oil

2.3 Measurement of hepatic lipids levels 3 RESULTS Liver lipids were extracted according to the method of 3.1 Effect of porpoise oil feeding on growth parameters in Folch et al.29）, and the concentrations of triglyceride, cho- OLETF rats lesterol, and phospholipid were measured by the methods There was no signifi cant difference in initial body weight, of Fletcher30）, Sperry and Webb31）, and Rouser et al.32）, re- fi nal body weight, food intake, liver and WAT（epididymal, spectively. perirenal, omental, and subcutaneous）weights between two groups（Table 2）. 2.4 Preparation of hepatic subcellular fractions A piece of liver was homogenized in six volumes of a 0.25 3.2 Effect of porpoise oil feeding on hepatic lipid levels in M sucrose solution that contained 1 mM EDTA in a 10 mM OLETF rats Tris-HCl buffer（pH 7.4）. After the nuclei fraction was pre- Although there was no signifi cant difference in hepatic cipitated, the supernatant was centrifuged at 10000 g for levels of cholesterol and phospholipid between two groups, 10 min at 4 ºC to obtain mitochondria fraction. The result- hepatic triglyceride level was signifi cantly lowered by the ing supernatant was recentrifuged at 125000 g for 60 min PO diet compared with the control diet（Fig. 1）. to precipitate microsomes, and the remaining supernatant was used as the cytosol fraction. The protein concentration 3.3 Effect of porpoise oil feeding on activities of hepatic was determined according to the method of Lowry et al.33）, enzymes related to triglyceride metabolism in OLETF with bovine serum albumin used as the standard. rats The activities of FAS, malic enzyme, and G6PDH, key 2.5 Assays of hepatic enzyme activity enzymes of fatty acid synthesis, were not changed by the The enzyme activities of phosphatidate phosphohydro- PO diet. The activity of PAP, a rate-limiting enzyme of tri- lase（PAP）in the microsome fraction34）, fatty acid synthase glyceride synthesis, was not changed by the PO diet. In ad- （FAS）in the cytosol fraction35）, glucose 6-phosphate dehy- dition, there was no signifi cant difference in the activities drogenase（G6PDH）in the cytosol fraction36）, malic enzyme of CPT, a rate-limiting enzyme of fatty acid β-oxidation in in the cytosol fraction37）, carnitine palmitoyltransferase the mitochondria, and peroxisomal β-oxidation between （CPT）in the mitochondria fraction38）, and peroxisomal two groups（Table 3）. β-oxidation39）were determined as described elsewhere. 3.4 Effect of porpoise oil feeding on serum lipids levels in 2.6 Statistical analysis OLETF rats All values are expressed as means±SEM. The signifi- Although there was no significant difference in serum cance of differences between means for two groups was levels of cholesterol, phospholipid between the groups, se- determined by Student’s t test. Differences were consid- rum triglyceride level was increased by the PO diet com- ered to be signifi cant at P < 0.05. pared with the control diet（Table 4）.

3.5 Effect of porpoise oil feeding on serum adipocytokine levels in OLETF rats Serum leptin level did not differ between two groups, Table 2 Effect of Porpoise Oil Feeding on Growth Parameters whereas serum adiponectin level was significantly in- in OLETF Rats Control group PO group Initial B.W. (g) 163 ± 4 162 ± 5 Final B.W. (g) 365 ± 8 355 ± 9 Food intake (g) 651 ± 14 651 ± 12 Liver weight (g/100g B.W.) 3.70 ± 0.07 3.77 ± 0.07 White adipose tissue weight (g/100 g B.W.) Epididymal 2.21 ± 0.12 2.25 ± 0.14 Fig. 1 Effect of Porpoise Oil Feeding on Hepatic Lipids Perirenal 3.03 0.12 2.92 0.10 ± ± Levels in OLETF Rats. Omental 1.61 ± 0.06 1.62 ± 0.07 Rats were fed control diet or PO diet for 4 weeks. Subcutaneous 3.92 ± 0.18 3.99 ± 0.15 Values are expressed as means ± standard error of Values are expressed as means ± standard error of six rats. six rats. Asterisk shows signiﬁ cant difference at B.W., body weight P<0.05. 301 J. oleo Sci. 59, (6) 299-305 (2010) B. Shirouchi , K. Nagao, K. Furuya et al.

Table 3 Effect of Porpoise Oil Feeding on Enzyme Activities Involved in Hepatic Triglyceride Metabolism in OLETF Rats Control group PO group

(nmol/min/mg protein) PAP 9.12 ± 0.41 8.88 ± 0.32 FAS 10.9 ± 0.6 11.6 ± 0.8 G6PDH 63.9 ± 4.5 57.5 ± 4.1 Malic enzyme 87.7 ± 4.8 89.2 ± 5.3 CPT 3.95 ± 0.25 4.30 ± 0.19 Peroxisomal β-oxidation 8.24 ± 0.97 8.37 ± 0.31 Values are expressed as means ± standard error of six rats. PAP, phosphatidate phosphohydrolase; FAS, fatty acid synthase; G6PDH, glucose 6-phosphate dehydrogenase; CPT, carnitine palmitoyltransferase.

Table 4 Effect of Porpoise Oil Feeding on Serum 4 DISCUSSION Lipids Levels in OLETF Rats We investigated the physiological functions of porpoise oil, which contains unique structured-lipids as combination Control group PO group of iso-C5:0 and ω3 PUFAs, in obese OLETF rats. After 4 (mg/dL) weeks of feeding, the PO diet significantly decreased he- Triglyceride 125 ± 16 198 ± 13 ＊ patic triglyceride levels in OLETF rats（Fig. 1）. To further Cholesterol 162 ± 6 152 ± 11 investigate the regulation of hepatic triglyceride metabolism, we analyzed the effect of porpoise oil feeding on ac- Phospholipid 242 ± 13 241 ± 11 tivities of enzymes related to triglyceride synthesis, fatty Values are expressed as means ± standard error of acid synthesis, and fatty acid β-oxidation. In the present six rats. study, the PO diet did not change activities of enzymes re- Asterisk shows signifi cant difference at P < 0.05. lated to hepatic lipid metabolism in OLETF rats（Table 3）. Although feeding of ω3 PUFAs（such as EPA and DHA）has been reported to suppress fatty acid synthesis and enhance fatty acid β-oxidation in the liver of rats18, 40）, the amounts of ω3 PUFAs in porpoise oil might not be enough to reveal benefi cial effects, such as suppression of fatty acid synthesis and/or enhancement of fatty acid β-oxidation, in the liver of obese rats. Additionally, there were signifi cant amount of unidentifi ed fatty acids in porpoise oil and their effects on hepatic enzyme activities could not be ignored. Further studies are necessary to clarify the effect of more purifi ed structured-lipid containing only iso-C5:0 and ω3 PUFAs on lipogenesis and lipolysis in the liver. In contrast with the alleviation of hepatic triglyceride accumulation, serum tri- Fig. 2 Effect of Porpoise Oil Feeding on Serum glyceride level was significantly higher in the PO group Adipocytokine Levels in OLETF Rats. than that in the control group. Microsomal triglyceride Rats were fed control diet or PO diet for 4 weeks. transfer protein（MTP）plays a crucial role in the assembly Values are expressed as means ± standard error of and secretion of apolipoprotein B containing lipoproteins, six rats. Asterisk shows signifi cant difference at in particular very low-density lipoprotein（VLDL）41, 42）. Al- P<0.05. though the extent and the direction（increase or decrease） of the liver lipoprotein synthesis and secretion during the development of fatty liver disease have been controver- creased by the PO diet compared with the control diet（Fig. sial43-45）, the enhancement of MTP activity may explain the 2）. decrease in the hepatic TG level concomitant with the increase in the serum TG level in PO diet-fed OLETF rats. 302 J. oleo Sci. 59, (6) 299-305 (2010) Physiological Functions of Porpoise Oil

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