13
Atherosclerosis, 84 (1990) 13-74 Elsevier Scientific Publishers Ireland, Ltd.
ATHERO
04535
Letter to the Editors
Apolipoprotein
AI mRNA levels in WHHL rabbits
Dear Editors,
Seftel [l] pointed out pertinent data on HDL deficiency in homozygous Watanabe heritable hyperlipidemic rabbits (WHHL), as related to a paper by Goldstein et al. [2]. To search for mechanisms involved in the HDL deficiency in homozygous WHHL, we carried out in vivo kinetic studies [3]. We obtained data to support the notion that the HDL deficiency was the result of a more rapid fractional catabolic rate and a reduced synthesis of HDL-apolipoprotein AI. We have now quantitated apo AI mRNA levels of the total RNA from liver and intestine using Northern blot analysis and an apo AI cDNA probe. Two male WHHL rabbits (2.6 and 3.0 kg body weight, over 10 months of age) were kindly provided by Dr. Y. Watanabe, Kobe University, Kobe, Japan. Normolipidemic male Japanese white (JW) rabbits (n = 2, 2.8 and 3.0 kg body wt., and of similar age to the WHHL rabbits) were obtained from Kyudo Co. Ltd., Fukuoka, Japan. Both groups of animals were fed a standard diet of RC-4 at 80 g/day per animal (Oriental Yeast Co., Ltd., Tokyo; crude fat and cholesterol contents 3.0 g and 2 mg per 100 g dry food, respectively). After an overnight fast each animal was anesthetized with xylazine and ketamine, and the liver and intestines were removed. Total RNA was isolated from 2 g (per animal) frozen rabbit liver or intestine in liquid N, by the guanidine thiocyanate method [4]. The RNA (30 pg) was electrophoresed on 1.5% agarose gels and transferred to a nitrocellulose membrane following electrophoresis. 32P-Labeled human apo AI cDNA probe was hybridized to the RNA and specific hybridization was visualized by autoradiography. Compared to findings in the normolipidemic JW rabbits (control group), the WHHL animals had 2-3-fold lower levels of intestinal apo AI mRNA (Fig. 1; another pair of rabbits showed exactly the same results), which was almost in line 0021-9150/90/$03.50
0 1990 Elsevier Scientific
Publishers
with the data of apo AI synthesis obtained from a previous HDL-apo AI kinetic in vivo study (18.14 f 3.75 mg/kg body weight per day for normolipidemic JW rabbits vs. 8.67 k 0.59 mg/kg body wt./day for WHHL rabbits) [3]. The mRNA of apo AI was absent in the liver of both groups. This finding confirms the impaired intracellular synthesis of apo AI in WHHL rabbits. A deficiency in the LDL receptor would affect gene expression of HDL-apo AI and may to some extent account for the progression of atherosclero-
Fig. 1. RNA gel-blot hybridization of apo AI to RNAs isolated from normal or WHHL rabbit intestine.
Ireland, Ltd.
74
sis in this rabbit model of familial hypercholesterolemia. References 1 Seftel, H.C., HDL cholesterol in familial hypercholesterolemia, N. Engl. J. Med., 310 (1984) 125.
Department of Internal Medicine, School of Medicine, Fukuoka University, 45-1, I-chome Nanakuma Jonan-ku, Fukuoka 814-01. Japan
(Received 22 May, 1990) (Revised, received 15 June, 1990) (Accepted 19 June, 1990)
2 Goldstein, J.L., Kita, T. and Brown, M.S., Defective lipoprotein receptors and atherosclerosis, N. Engl. J. Med., 309 (1983) 288. 3 Saku, K., Yamamoto, K., Sakai, T., Yanagida, T., Hidalca, K., Sasaki, J. and Arakawa, K., Kinetics of HDL-apo A-I in the WHHL rabbit, an animal model of familial hypercholesterolemia, Atherosclerosis, 79 (1989) 225. 4 Chirgwin, J.M., Przybyla, A.E., MacDonald, R.J. and Rutter, W.J., Biochemistry, 18 (1979) 5294.
Keijiro Saku, Ryoko Harada, Kyosuku Yamamoto, Hong Ying, Iwata Ozaki and Kikuo Arakawa
Atherosclerosis, 84 (1990) 13-74 Elsevier Scientific Publishers Ireland, Ltd.
ATHERO
04535
Letter to the Editors
Apolipoprotein
AI mRNA levels in WHHL rabbits
Dear Editors,
Seftel [l] pointed out pertinent data on HDL deficiency in homozygous Watanabe heritable hyperlipidemic rabbits (WHHL), as related to a paper by Goldstein et al. [2]. To search for mechanisms involved in the HDL deficiency in homozygous WHHL, we carried out in vivo kinetic studies [3]. We obtained data to support the notion that the HDL deficiency was the result of a more rapid fractional catabolic rate and a reduced synthesis of HDL-apolipoprotein AI. We have now quantitated apo AI mRNA levels of the total RNA from liver and intestine using Northern blot analysis and an apo AI cDNA probe. Two male WHHL rabbits (2.6 and 3.0 kg body weight, over 10 months of age) were kindly provided by Dr. Y. Watanabe, Kobe University, Kobe, Japan. Normolipidemic male Japanese white (JW) rabbits (n = 2, 2.8 and 3.0 kg body wt., and of similar age to the WHHL rabbits) were obtained from Kyudo Co. Ltd., Fukuoka, Japan. Both groups of animals were fed a standard diet of RC-4 at 80 g/day per animal (Oriental Yeast Co., Ltd., Tokyo; crude fat and cholesterol contents 3.0 g and 2 mg per 100 g dry food, respectively). After an overnight fast each animal was anesthetized with xylazine and ketamine, and the liver and intestines were removed. Total RNA was isolated from 2 g (per animal) frozen rabbit liver or intestine in liquid N, by the guanidine thiocyanate method [4]. The RNA (30 pg) was electrophoresed on 1.5% agarose gels and transferred to a nitrocellulose membrane following electrophoresis. 32P-Labeled human apo AI cDNA probe was hybridized to the RNA and specific hybridization was visualized by autoradiography. Compared to findings in the normolipidemic JW rabbits (control group), the WHHL animals had 2-3-fold lower levels of intestinal apo AI mRNA (Fig. 1; another pair of rabbits showed exactly the same results), which was almost in line 0021-9150/90/$03.50
0 1990 Elsevier Scientific
Publishers
with the data of apo AI synthesis obtained from a previous HDL-apo AI kinetic in vivo study (18.14 f 3.75 mg/kg body weight per day for normolipidemic JW rabbits vs. 8.67 k 0.59 mg/kg body wt./day for WHHL rabbits) [3]. The mRNA of apo AI was absent in the liver of both groups. This finding confirms the impaired intracellular synthesis of apo AI in WHHL rabbits. A deficiency in the LDL receptor would affect gene expression of HDL-apo AI and may to some extent account for the progression of atherosclero-
Fig. 1. RNA gel-blot hybridization of apo AI to RNAs isolated from normal or WHHL rabbit intestine.
Ireland, Ltd.
74
sis in this rabbit model of familial hypercholesterolemia. References 1 Seftel, H.C., HDL cholesterol in familial hypercholesterolemia, N. Engl. J. Med., 310 (1984) 125.
Department of Internal Medicine, School of Medicine, Fukuoka University, 45-1, I-chome Nanakuma Jonan-ku, Fukuoka 814-01. Japan
(Received 22 May, 1990) (Revised, received 15 June, 1990) (Accepted 19 June, 1990)
2 Goldstein, J.L., Kita, T. and Brown, M.S., Defective lipoprotein receptors and atherosclerosis, N. Engl. J. Med., 309 (1983) 288. 3 Saku, K., Yamamoto, K., Sakai, T., Yanagida, T., Hidalca, K., Sasaki, J. and Arakawa, K., Kinetics of HDL-apo A-I in the WHHL rabbit, an animal model of familial hypercholesterolemia, Atherosclerosis, 79 (1989) 225. 4 Chirgwin, J.M., Przybyla, A.E., MacDonald, R.J. and Rutter, W.J., Biochemistry, 18 (1979) 5294.
Keijiro Saku, Ryoko Harada, Kyosuku Yamamoto, Hong Ying, Iwata Ozaki and Kikuo Arakawa
