帝塚山学院・人間科学部研究年報１９☆／７．福田

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1 Effects of Diet Restriction on Indices of Glucose and Lipid Metabolism of Rats Hitomi Fukuda, Tomoe Hirakawa, Nobuko Iritani Formerly of Tezukayama Gakuin University acetate TAG : :00 6:00 9:00 0:00 6: Abstract In a preliminary experiment, triacylglycerol synthesis from labeled acetate was decreased with decreasing diet quantity in rats fed of ad libitum intake of fat free diet, suggesting that the 80 diet may be possible restriction. The effects of diet restriction on glucose and lipid metabolism were measured at 3 h intervals beginning at 18 : 00 in rats given diet at 17 : 00, after feeding ad libitum control or 80 feeding of control for 2 weeks. By the diet restriction, the plasma and liver triacylglycerols were significantly decreased and the weights of visceral adipose tissues were significantly decreased with decrease of body weight increment. The stomach contents in the diet restriction group were

2 greater than in the control until 3 : 00 the next morning and then lesser after 9 : 00. In the restricted group, the plasma free fatty acid levels increased and the insulin levels decreased during diet depletion. The hepatic glycogen reached a high peak similar to the control group in 7 h after feeding but 6 h earlier than in the control. The mrna concentrations of muscle Glut 4 and liver insulin receptors were not significantly different between the two groups. The diurnal changes were more greatly observed in the diet restriction rather than in the control. It is suggested that the 80 diet was not enough to keep health during diet depletion. Keywords : Rat, Diet Restriction, Glucose and Lipid Metabolism kcal 1895 kcal kcal, 1662 kcal 3 4 mrna 1 TAG C NEFAC E- 125 I RAT INSULIN RIA KIT Millipore Corporation Linco Massachusetts, U.S.A 14 : 0, 1.1 ; 16 : 0, 30.4 ; 18 : 0, 17.9 ; 18 : 1, 41.2 ; 18 : 2, C Acetic acid, sodium salt MBq/mmol Moravek Biochemicals Inc. Brea, CA,

3 USA g/100 g g/100 g 2 4 Wistar SLC 3 17 : am-8pm g 74 kbq /0.1 ml acetate 2 TAG TAG TAG acetate ,000 xg Wistar : : M 105,000 xg

4 3 Fatty acid synthase Fatty acid synthase FAS, EC Hsu ,000 xg mu/mg protein 4 RNA mrna RNA guanidium thiocyanate-phenol-chloroform 9 RNA 80 FAS Uncuppling protein UCP Insulin Receptor Glucose transporter 4 GLUT4 Taqman PCR Life Technology Co., Insulinreceptor : Rn _ml, 140 bp ; GLUT4 : Rn _ml, 89 bp ; GAPDH : Rn _gl, 174 bp RT-PCR mrna GAPDH 5 Folch 12 TAG 7 TAG Good 13 6 Two-way ANOVA SPSS Ver 21, IBM Turkey Table g g g g, g 70 9g Table

Table 1 Effect of diet quantity restriction on body weight gain, liver and epidydimal adipose tissue weight for 4 days feeding Diet quantity 100 90 80 70 Diet quantity g/100 g bw/day 14.5 0.66 a 13.

5 Table 1 Effect of diet quantity restriction on body weight gain, liver and epidydimal adipose tissue weight for 4 days feeding Diet quantity Diet quantity g/100 g bw/day a b c d Final body weight g a ab bc bc Body weight gain g a b c c Liver g/100 g bw Adipose tissue g/100 g bw a b b b The body weights at the start of feeding were not significantly different in the 100 control, 90, 80 and 70 feeding. Mean SD n 6. Means with different superscript letters are significantly different at p g 100 g 1.2 acetate acetate TAG Fig TAG TAG 90 Fig. 1 Effects of diet quantity restriction on incorporation of labeled acetate into total lipids and TAG in the liver, plasma and adipose tissue after the intravenous injection Male Wistar rats, 4-5 weeks old, were fed a fat-free diet 100, 90, 80 or 70 feeding of ad libitum 100, for4 d. The rats were intravenously injected MBq 1-14 C -acetate /kg bw at 9.00 h, and then killed 2 h after the labeled acetate injection. Labeled acetate incorporation into total and total lipids in liver, plasma and epididymal adipose tissue for 2 h after the injection were measured. Total, total lipid and TAG radioactivities are shown dpm/ gorml. Mean SD n 4. One-way ANOVA for food quantity : p in liver, plasma and adipose tissue.

60 80 70 20 70 20 TAG 90 80 70 50 TAG Fig. 2 TAG g 80 70 Table 1 90 80 70 56 24 15 90 TAG 80 80 Fig.

6 TAG TAG Fig. 2 TAG g Table TAG Fig. 2 Effects of diet quantity restriction on liver, plasma and adipose tissue TAG levels The rats were the same in Fig.1. Mean SD n 4. One-way ANOVA for food quantity : p in liver and plasma : 00 9:00 17 : 00 Fig. 3 16

7 Fig. 3 Effects of diet quantity restriction on diet intake and stomach contents Male Wistar rats, 8 wk old, were fed ad libitum intake 100, control or 80 feeding of a control for 2 wk. The rats were maintained on a 12-h-light : -dark cycle lights on at 0800 h. The upper and middle figures show the diet intake g/100 g bw/h of control and 80 feeding, respectively. The stomach contents g/ 100 g bw are shown at each time. Mean SD n 6. Two-way ANOVA for stomach contents : diet quantity D, p ; time T, p ; DxT,p Rats were the same to those shown in Fig : :00 9: :00 Table 2 Effect of diet quantity restriction on body weight gain, liver and adipose tissue weight for 2 weeks feeding Diet quantity Final body weight g * Body weight gain g * Liver g/100 g bw * Epidydimal adipose tissue g/100 g bw * Perirenal adipose tissue g/100 g bw * Mesenteric adipose tissue g/100 g bw * The body weights at the start of the experiment were not significantly different in the control 100 and 80 feeding. Mean SD n 6. *Significantly different from control group, p

8 13 6:00 3: :00 15 : g g g g Table FFA :00 6:00 Fig : :00 18 : 00 3 Fig. 4 Effects of diet quantity restriction on plasma glucose, insulin and free fatty acids FFA levels Mean SD n 6. Two-way ANOVA for glucose : D, p 0.01 ; T, p 0.05, for plasma insulin : D, p ; T, p 0.05, for plasma free fatty acids : T, p ; DxT,p 0.01.

9 FFA 16 9: :00 FFA 2.3 TAG TAG 16 9:00 TAG 9:00 12 : 00 0:00 6:00 TAG 50 Fig. 5 Fig. 5 Effects of diet quantity restriction on plasma and liver TAG levels Mean SD n 6. Two-way ANOVA for plasma TAG : D, p 0.001, D x T, p 0.05, for liver TAG : D, p ; T : : :00 9:00 6 Fig Acetyl-CoA carboxylase, Fatty acid synthase, Malic enzyme, Glucose-6-

Fig. 6 Effects of diet quantity restriction on liver glycogen levels Mean SD n 6. Two-way ANOVA for liver glycogen : D, p 0.001 ; T, p 0.001 ; DxT,p 0.001. Fig.

10 Fig. 6 Effects of diet quantity restriction on liver glycogen levels Mean SD n 6. Two-way ANOVA for liver glycogen : D, p ; T, p ; DxT,p Fig. 7 Effects of diet quantity restriction on FAS mrna concentrations and enzyme activities The mrna concentrations were normalized to the values at 9 : 00 of the control rats. The enzyme activities are shown as mu/mg protein. Mean SD n 6. Two-way ANOVA for mrna concentrations : T, p phosphate dehydrogenase, ATP citrate lyase fatty acid synthase FAS FAS mrna 8h FAS FAS mrna :00 3:00 Fig. 7 mrna :00 12 : 00 FAS mrna

2.6 UCP 1, 2, 3 mrna UCP2 mrna 4 GLUT4 1 18 : 00 Fig. 8 mrna 7 16 0:00 9:00 1 18 : 00 Fig.

11 2.6 UCP 1, 2, 3 mrna UCP2 mrna 4 GLUT : 00 Fig. 8 mrna :00 9: : 00 Fig. 8 Effects of diet quantity restriction on GLUT4 mrna concentrations of muscle and insulin receptor mrna concentrations of liver The mrna concentrations were normalized to the values at 9 : 00 of the control rats. Two-way ANOVA for GLUT4 mrna : T, p 0.05, for insulin receptor mrna : T, p mrna Table g TAG Fig. 1 TAG 90

12 Fig TAG : : 00 0:00 90 Fig. 3 9:00 FFA Fig. 4 9:00 3:00 17 : Sakamoto 22 FFA TAG :00 9:00 17 : 00 3: : 00 Fig. 6 1 mrna 23 FAS mrna mrna 10 Fig. 7 mrna 6 mrna mrna acetyl- CoA citrate malonyl-coa Km Ka 25

13 p p Sherman H, Frumin I, Gutman R, Chapnik N, Lorentz A, Meylan J, le Coutre J, Froy O 2011 Long-term restricted feeding alters circadian expression and reduces the level of inflammatory and disease markers. JCell Mol Med 15 : Folia Pharmacol. Jpn. 137 : Reeves PG, Nielsen FH, Fahey GC Jr 1993 Purified diets for laboratory rodents : Final report of the American Institute of Nutrition and Hoc Writing Committee on the reformation of the AIN-76 A rodent Diet. JNutr 123 : Iritani N, Hirakawa T, Fukuda H, Katsukawa M, Kouno M 2014 Comparison of labeled acetate and glucose incorporations into lipids in the liver and adipose tissue after intravenous injection in rats. J Nutr Sci Vitaminol Tokyo 60 : Kimura T, Fukuda H, Iritani N 2005 Labeled acetate incorporation into lipids and lipid elimination after oral administration in rat liver and adipose tissue. J Nutr Sci Vitaminol Tokyo 51 : Hsu RY, Butterworth PHW, Porte JW 1969 Pigeon liver fatty acid synthetase. Methods Enzymol 14 : Chomczynski P, Sacchi N 1987 Single-step method of RNA isolation by acid guanidium thiocyanate-phenolchloroform extraction. Anal Biochem 163 : Katsurada A, Iritani N, Fukuda H, Matsumura Y, Nishimoto N, Noguchi T, Tanaka T 1990 Effects of nutrients and hormones on transcriptional and post-transcriptional regulation of fatty acid synthase in rat liver. Eur J Biochem 190 : Iritani N, Sugimoto T, Fukuda H, Tomoe K 2002 Changes in UCP family expressions in rat tissues due to diet and aging. J Nutr Sci Vitaminol Tokyo 48 : Folch J, Lees M, Sloane-Stanley GH 1957 A simple method for isolation and purification of total lipids from animal tissues. JBiolChem226 : Good CA, Kramer H, Somogy M 1933 The determination of glycogen. JBiolChem100 : Snedecor GW, Cochran WG 1967 Statistical Methods. p Iowa State University Press, Ames, IA. Kohsaka A, Laposky AD, Ramsey KM, Estrada C, Joshu C, Kobayashi Y, Turek FW, Bass J 2007 High-fat diet disrupts behavioral and molecular circadian rhythms in mice. Cell Metabo 6 : Iritani N 1992 Nutritional and hormonal regulation of lipogenic-enzyme gene expression in rat liver. A review. Eur J Biochem 205 : Katsurada A, Iritani N, Fukuda H, Noguchi T, Tanaka T 1987 Influence of diet on the transcriptional and

14 post-transcriptional regulation of malic enzyme induction in the rat liver. Eur J Biochem 168 : Katsurada A, Iritani N, Fukuda H, Matsumura Y, Noguchi T, Tanaka T 1989 Effects of nutrients and insulin on transcriptional and post-transcriptional regulation of glucose-6-phosphate dehydrogenase synthesis in rat liver. Biochim Biophys Acta 1006 : Katsurada A, Iritani N, Fukuda H, Matsumura Y, Nishimoto N, Noguchi T, Tanaka T 1990 Effects of nutrients and hormones on transcriptional and post-transcriptional regulation of acetyl-coa carboxylase in rat liver. Eur J Biochem 190 : Fukuda H, Katsurada A, Iritani N 1992 Effects of nutrients and hormones on gene expression of ATP citratelyase in rat liver. Eur J Biochem 209 : Hatori M, Vollmers C, Zarrinpar A, DiTacchio L, Bushong EA, Gill S, Leblanc M, Chaix A, Joens M, Fitzpatrick JA, Ellisman MH, Panda S 2012 Time-restricted feeding without reducing caloric intake prevents metabolic diseases in mice fed a high-fat diet. Cell Metab 15 : Sakamoto S, Muto T, Yokota M, Ishimura N, Niwa Y, Harada N, Okada K, Nakaya Y 2000 Comparison between short-term food restriction and exercise on whole body glucose disposal in high-fat rats. J Med Invest 47 : Fukuda H, Iritani N 1991 Diurnal variations of lipogenic enzyme mrna quantities in rat liver. Biochim Biophys Acta 1086 : Iritani N, Nishimoto N, Katsurada A, Fukuda H 1992 Regulation of hepatic lipogenic enzyme gene expression by diet quantity in rats fed a fat-free, high carbohydrate diet. J. Nutr 122 : Fukuda H, Katsurada A, Iritani N 1985 Diurnal variations of lipogenic enzymes, their substrate and effector levels, and lipogenesis from tritiated water in rat liver. Biochim Biophys Acta 835 : Yanagihara H, Ando H, Hayashi Y, Obi Y, Fujimura A 2006 High-fat feeding exerts minimal effects on rhythmic mrna expression of clock genes in mouse peripheral tissues. Chronobiol Int 23 :

Table 1 Components of corn dietary fibers

Table 1 Components of corn dietary fibers Effects of Purified Corn Dietary Fiber on Blood Glucose, and Serum and Liver Lipids in Normal Rats and Mice Sachie Ikegami *1, Saeko Ohsawa *1, Shouko Machida *2 and Akiko Hada *2 The National Institute