892320B002212

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2 NSC B TNF-α ADD-1 (TG) Obesity is a result of increased number of adipocytes and/or amount of 3T3-L1 triacylglycerol (TG) in adipocytes. Accumulation of TG in adipocytes may lead to resistance to insulin, and moreover, TNF-α systematic metabolic disorders. In this study, we intended to investigate the expression of 3T3-L1 TNF-α and insulin sensitivity in adipocytes that has accumulated large amount of TG. (chylomicron) However, we were unable to measure 3T3-L1 TNF-α mrna accurately, because of very low expression of TNF-α. Ιn the experiment we found that chylomicron can be used to deliver fat into 3T3-L1 adipocytes. Although ADD-1/SREBP-1c it is known TG synthesis is stimulated by 3T3-L1 chylomicron through acylation stimulating ADD-1/SREBP-1c mrna protein(asp), the mechanism is not fully 68 kd ADD-1/SREBP-1c understood. We found that chylomicron (OB) increased both ADD-1/SREBP-1c mrna mrna and protein in 3T3-L1 adipocytes. In addition, C57Bl/6J OB mrna was also increased by chylomicron. In an animal study, we observed weight of epididymal fat pad was EPA 3T3-L1 significantly lower in mice fed a fish oil diet EPA than that of mice a fed soybean oil or a 68 kd ADD-1/SREBP-1c coconut oil diet. We then tested the effect of EPA, a major effective component of fish oil, ADD-1/SREBP-1c OB on ADD-1/SREBP-1c in 3T3-L1. The result showed that the 68 kd ADD-1/SREBP-1c protein was downregulated by EPA after 24 h of treatment. Our results suggest that chylomicron may regulate TG synthesis and OB gene expression through 2

3 ADD-1/SREBP-1c. It is very possible that EPA may also regulate TG synthesis through a similar pathway. : obesity, adipocyte, triglyceride, Leptin, TNF-α, ADD- C57Bl/6J mice apo C-III mrna Steppan resistin resistin PPAR-γ agonist resiglitazone PPAR-γ 1 resistin (insulin resistance) (Type II diabetes mellitus ) 2 (obesity associated db/db db/db insulin resistance) (metabolic study) db/db 6-7 TG 8-12 EPA DHA db/db (ob/ob, db/db) 13, ,17 23 rodents 18 EPA & DHA β-oxidation TG VLDL 19,20 C57BL/6J resistin Chylomicron VLDL-TG 8% 12% lipoprotein lipase LPL 24% C57BL/6J fatty acid LPL apo C-II apo C-III 12 TG fibrates PPAR-α apo C-III Table PPAR-α acyl-coa oxidase and cytochrome 8% 12% P450-4A2 resistin mrna 35% apo C-III 40% Figure 2-1 resistin mrna 3

4 mrna 300 µm EPA apo C-III bind to 100 µm albumin 50~100 µm 3T3-L1 0~48 EPA Hep G2 24 apo 300 µm EPA resistin mrna EPA resistin mrna PPAR-γ (Figure3-4) apo C-III mrna 19.5 C18 C57BL/6J coconut oil, soybean oil fish oil 6 C20:4(n-6), C20:5(n-3) (fasted C22:6(n-3) Hep G2 apo C-III group) 3 insulin refed group apo C-III 6 12 db/db resistin apo C-III mouse resistin mrna apo C-III genomic DNA resistin LPS Figure 3-1 & 3-2 rats adipose tissue resistin apo Nature C-III mrna resistin resistin apo C-III LPL resistin TG VLDL apo C-III TG EPA (Hep G2) apo C-III C-III (Figure3-3) insulin DHA apo C-III 0, C18:1, C18:2(n-6), C18:3(n-3), 4

5 7. Thomas CD, Peters JC, Reed GW, 1., Abumrad NN, Sun M, Hill JO (1992)Nutrient balance and energy expenditure during ad 2. Reaven GM (1988) Role of libitum feeding of high-fat and insulin resistance in human high-carbohydrate diets in disease. Diabetes 37: humans. Am J Clin Nutr 3. Marshall JA, Hamman RF, Baxter 55(5): J (1991) High-fat, lowcarbohydrate diet and the 8. Ikemoto S, Takahashi M, Tsunoda N, Maruyama K, Itakura H. & Ezaki etiology of non-insulindependent diabetes mellitus: the O. (1996) High-fat diet induced hyperglycemia and obesity in mice: San Luis Valley Diabetes Study. differential effects of dietary Am J Epidemiol 134: oils. Metabolism 45: Stern MP (1991)Primary prevention of type II diabetes mellitus. Diabetes Care 14(5): Storlien LH, James DE, Burleigh KM, Chisholm DJ, Kraegen EW (1986)Fat feeding causes widespread in vivo insulin resistance, decreased energy expenditure, and obesity in rats. Am J Physiol 251:E Flatt JP (1987) The difference in the storage capacities for carbohydrate and for fat, and its implications in the regulation of body weight. Ann N Y Acad Sci 499: Parker DR, Weiss ST, Troisi R, Cassano PA, Vokonas PS & Landsberg L. (1993) Relationship of dietary saturated fatty acids and body habitus to serum insulin concentrations: the Nomative Aging Study. Am J Clin Nutr 58: Pan DA & Strolien LH (1993) Dietary lipid profile is a determinant of tissue phospholipid fatty acid composition and rate of weight gain in rats. J Nutr 123: Pan DA, Hulbert AJ, & Storlien LH (1994) Dietary fat, membrane 5

6 phospholipids and obesity. J Nutr l24:l Rustan AC, Nenseter MS. & Drevon CA.(1997) Omega-3 md omega-6 fatty acids in the insulin resistance syndrome. Lipid and lipoprotein metabolism and atherosclerosis. Ann N Y Acad Sci 827: Cunnane SC, McAdoo KR & Horrobin DF. (1986) n-3 Essential fatty acids decrease weight gain in genetically obese mice. Br J Nutr 56: Miura T, Ohnishi Y, Takagi S, Sawamura M, Yasuda N, Ishida H, Tanigawa K, Yamori Y & Seino Y (1997) A comparative study of high-fat diet containing fish oil or lard on blood glucose in genetically diabetic (db/db)mice. J Nutr Sci Vitaminol (Tokyo) 43: Storlien LH, Kraegen EW, Chisholm DJ, Ford GL, Bruce DG & Pascoe WS. (1987) Fish oil prevents insulin resistance induced by high-fat feeding in rats. Science 237: Heine RJ, Mulder C, Popp-Snijders C, Van Der Meer J. & Van Der Veen EA. (1989) Linoleic-acid-enriched diet: long term effcts on serum lipoprotein and apolipoprotein concentrations and insulin sensitiity in noninsulindependent diabetic patients. Am J Clin Nutr 49: Luo J, Rizkalla SW, Boillot J, Alamowitch C, Chaib H, Bruzzo F, Desplanque N, Dalix AM, Durand G & Slama G (1996) Dietary (n-3) polyunsaturated fatty acids improve adipocyte insulin action and glucose metabolism in insulin-resistant rats: relation to membrane fatty acids. J Nutr 126: Taskinen MR (1995) Insulin resistance and lipoprotein metabolism. Curr Opin Lipidol 6: Halminski, M. A., Marsh, J. B., and Harrison, E. H. (1991) Differential effects of fish oil, safflower oil and palm oil on fatty acid oxidation and glycerolipid synthesis in rat liver. J. Nutr. 121: Nossen, J. O., Rustan, A. C., Gloppestad, S. H., Malbakken, S., and Drevon, C. A. (1986) Eicosapentaenoic acid inhibits 6

7 synthesis and secretion of triacylglycerols by cultured rat hepatocytes. Biochim. Biophys. Acta 879: Hertz R, Bishara-Shieban J, Bar-Tana J 1995 Mode of action of peroxisome proliferators as hypolipidemic drugs. Suppression of apolipoprotein C-III. J Biol Chem. 270: Steppan CM, Bailey ST, Bhat S, Brown EJ, Banerjee RR, Wright CM, Patel HR, Ahima RS, Lazar MA The hormone resistin links obesity to diabetes. Nature 409: Prince MJ & Deeg MA 1997 Do n-3 fatty acids improve glucose tolerance and lipemia in diabetics? Curr Opin Lipidol 8:

8 Table 1-1. Effects of fish oil on body weight and plasma lipid and glucose concentration in db/db mice. Soybean Oil Fish Oil Initial Body Weight (g) Finial Body Weight (g) Weight Gain (g) (+ 4.9 %) (- 4.3%)* Feed Efficiency * Plasma Cholesterol (mg/dl) Plasma Triacylglycerol (mg/dl) * Plasma Glucose (mg/dl) * 1 Values are Mean + SD. * p<0.05 as compared with soybean oil group. 8

9 Table 2-1. Effects of dietary fish oil on body weight, epididymal adipose tissue weight and plasma TG in C57Bl/6J mice. 5% SO 24% SO 16% SO 12% SO +8% FO + 12% FO Number of mice Initial weight g 20.5± ± ± ±1.2 Final weight g 29.6± ± ± ±2.4 Feed efficiency ± Adipose weight g 0.30± ± ± ±0.06 TG(mg/dL) 55.0± ± ± ±5.3 Values are mean+sd. SO: soybean oil; FO, fish oil; TG, triacylglycerol. 9

10 Figure 2-1. Dietary fish oil down-regulate resistin mrna expression. Male C57BL/6J mice were fed 5 soybean oil (5% SO), 24 soybean oil(24% SO), 16 soybean oil + 8% fish oil(16% SO+ 8% FO) or 12% soybean oil + 12% fish oil(12% SO+ 12% FO) for 12 weeks. 10g of adipose total RNA was analyzed for resistin mrna abundances by Northern blot analysis. Figure 2-2. EPA down-regulate resistin mrna expression in 3T3-L1 adipocytes in a time-dependent manner. Differentiated 3T3-L1 adipocytes were treated with 300 µm EPA for 0 to 48 h. Resistin mrna was determined by Northern blot analysis. 10

11 Table 3-1. Effects of dietary fish oil on body weight, epididymal adipose tissue weight and plasma TG in C57Bl/6J mice. Coconut Oil Soybean OilFish Oil Number of mice Initial weight g 19.4± ± ±0.2 Final weight g 28.0± ± ±0.2 Liver Weight (g) Adipose Weight (g) * Table 3-2. Effects of dietary fat on plasma triglyceride and cholesterol concentrations in fasted and refed states. Coconut Oil Soybean Oil Fish Oil Fasted Refed Fasted Refed Fasted Refed Triglyceride b a b a c b (mg/dl) 74% 68% 28% Cholesterol b a cd b d c (mg/dl) Values are mean+sd of 6-8 mice. Values not sharing a same superscript letter in the same row are significantly different from one another at p<

12 Apo CIII CO SO FO Fasted Refed Fasted Refed Fasted Refed GAPDH Fig Effects of dietary fat and fasting/refeeding on hepatic Apo C-III mrna level of C57BL/6J mice. Weaning male C57BL/6J mice were fed 20 coconut oil(co), 20 soybean oil(so), or 20 fish oil(fo) for 6 weeks. 10g of hepatic total RNA was analyzed for Apo CIII mrna abundances by Northern blot analysis. * p< 0.05, as compared with the fasted coconut oil group; ** p<0.05, as compared with fasted fish oil group. 12

13 CO SO FO Fasted Refed Fasted Refed Fasted Refed ApoCIII GAPDH Fig Effects of dietary fat and fasting/refeeding on intestinal Apo C-III mrna level of C57BL/6J mice. Weaning male C57BL/6J mice were fed 20 coconut oil(co), 20 soybean oil(so), or 20 fish oil(fo) for 6 weeks. 10g of intestinal total RNA was analyzed for Apo CIII mrna abundances by Northern blot analysis. * p< 0.05, as compared with the fasted coconut oil group; ** p<0.05, as compared with corresponding fasted group. 13

14 Figure 3-3. EPA down-regulate Apo C-III mrna expression dose-dependently in Hep G2 cells. 0, 50, 75 or 100 M EPA was added to the culture medium of Hep G2 cells for 24 h. Apo C-III mrna was detedted by Northern blot analysis and relative level was shown after corrected with GAPDH. Values are mean + SD of 3 independent experiments. * p<0.05, as compared with control. 14

15 Figure 3-4. EPA and insulin down-regulate Apo C-III mrna expression in Hep G2 cells additively. 75 M EPA, 1 g/ml insulin or 75 M EPA+1 g/ml insulin was added to the culture medium of Hep G2 cells for 24 h. Apo C-III mrna was detedted by Northern blot analysis and relative level was shown after corrected with GAPDH. Values are mean + SD of 3 independent experiments. * p<0.05, as compared with control. 15

中草药 Chinese Traditional and Herbal Drugs 第 46 卷第 9 期 2015 年 5 月 1339 非酒精性脂肪性肝病 (nonalcoholic fatty liver disease,nafld) 是一种与胰岛素抵抗遗

中草药 Chinese Traditional and Herbal Drugs 第 46 卷第 9 期 2015 年 5 月 1339 非酒精性脂肪性肝病 (nonalcoholic fatty liver disease,nafld) 是一种与胰岛素抵抗遗 1338 中草药 Chinese Traditional and Herbal Drugs 第 46 卷第 9 期 2015 年 5 月九味肝泰胶囊对高脂饮食诱导大鼠非酒精性脂肪肝的治疗作用陈菲 1 2,, 艾国 3, 盛柳青 1, 麻佳蕾 1, 黄正明 1. 金华职业技术学院医学院, 浙江金华