Transgenic Mini Rat Strain as a Tool for Studying Aging and Calorie Restriction

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Isao Shimokawa

Mini rats, a transgenic strain of rats whose somatotropic axis was suppressed by overexpression of the antisense growth hormone gene, were shown to live longer than nontransgenic wild-type rats (—/—), when heterozygous for the transgene (tg/—); homozygous (tg/tg) rats died slightly earlier due to neoplastic causes. As observed in (tg/—) rats, moderate suppression of the somatotropic axis produced some phenotypes similar to those in (—/—) rats subjected to calorie restriction (CR), a well-known experimental intervention favoring longevity in animals. Thus, comparative studies using (tg/—) rats with the CR paradigm will help us understand the role of the somatotropic axis in regulation of lifespan and aging. Furthermore, the level of suppression of the somato-tropic axis in (tg/—) rats was not as severe as in other mice models, and thus, experiments can be performed within physiological ranges.

Introduction

In the last decade, since Ames dwarf mice with spontaneous mutation of the prop-1 gene were reported to live longer than their wild-type counterparts (Brown-Borg et al., 1996), over 10 rodent longevity models, in which a single gene has been spontaneously mutated or genetically engineered, have been reported (Miskin and Masos, 1997; Migliaccio et al., 1999; Coschigano et al., 2000; Flurkey et al., 2001; Mitsui et al., 2002; Shimokawa et al., 2002; Bluher et al., 2003; Holzenberger et al., 2003). Many of the mice used show a reduction in growth hormone (GH)-insulin-like growth factor (IGF)-1 signaling or insulin signaling (Liang et al., 2003). The importance of these models can be stressed in two ways. First, the DAF-2- AGE-1 pathway in nematodes increases lifespans when the signal input is reduced (Hekimi and Guarente 2003). These two molecules are orthologues of mammalian insulin or IGF receptor (Kimura et al., 1997) and phosphatidylinositol 3-kinase (PI3K; (Morris et al., 1996)), respectively. In fruit flies, mutation of the ''Chico'' gene, an orthologue of mammalian insulin receptor substrates, in the insulin-like pathway is also known to result in an extended lifespan (Clancy et al., 2001). These findings suggest that insulin or IGF-1 signaling is an evolutionarily conserved pathway that controls longevity in animals. Second, calorie restriction (CR), a well-known experimental intervention for lifespan extension in a wide range of organisms, also suppresses GH-IGF-1/insulin signaling (Masoro, 2003), suggesting that CR increases animal lifespans partly through a reduction in the signaling pathway (Shimokawa et al., 2003).

In the following chapter, a transgenic strain of mini rats whose somatotropic axis was suppressed by overexpression of the antisense GH gene (Matsumoto et al., 1993) is described and compared with CR rats in terms of lifespan, pathology, and selected biomarkers potentially related to the effect of CR.

Mini Rats and Their Husbandry

The transgenic rats were produced from founders created by introducing fusion genes into rat embryos (Matsumoto et al., 1993); their genetic background was Jcl: Wistar (Japan Clea, Inc., Tokyo, Japan). The transgene consisted of four copies of thyroid hormone response elements, rat GH promoter, and antisense cDNA sequences for rat GH. Transgenic offspring expressed the rat GH antisense transgene in the pituitary gland, and exhibited dwarfism as early as 3 weeks of age. Antisense GH-mRNA expression was detected by RT-PCR in the pituitary gland, spleen, and thymus in transgenic rats at 6 months of age, but not in the lungs, liver, heart, kidneys or testis (Shimokawa et al., 2002).

Male and female transgenic rats showed a reduced growth rate but almost normal reproduction function, although maturation seemed to be slightly delayed and the fecundity in female transgenic rats was slightly lower than in control female Wistar rats.

F1 hybrid rats (Jcl:Wistar-TgN (ARGHGEN)1Nts x Jcl:Wistar) were also generated at our laboratory animal center to moderate the reduced level of suppression

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of the somatotropic axis. The experimental animals were referred to as (tg/tg), (tg/—), and (—/—) with respect to the presence of the transgene.

At 4 weeks of age, weanling male rats were transferred to a barrier facility (temperature, 22 to 25°C; 12-h light/ dark cycle), housed separately, and maintained under specific pathogen-free (SPF) conditions. The procedure for monitoring the SPF status was fully described elsewhere (Shimokawa et al., 2002) and has been maintained since 1997 when the rat colony was established.

Rats were provided with a CR-LPF diet (Oriental Yeast, Tsukuba, Japan) based on the formula of Charles River (CRF-1), but with a protein fraction of 18.2%; the original CRF-1 diet contains 22.6% protein on a weight basis. All rats were fed a CR-LPF diet and water ad libitum (AL) after weanling. At 6 weeks of age, rats were divided into two diet groups: one group (AL) continued to receive food ad libitum, while the other (CR) was provided every other day with 140% of the mean daily food intake of the AL group in each rat group 30 min before the lights were turned off. This regimen successively restricted the food intake of the CR rats by 30% that of the AL group during the entire experimental period. However, it also yielded a 2-day cycle in the pattern of food intake in the CR group, with most of the allotted food being ingested within the first 24-h. When the food intake pattern confounded serum insulin and other hormones, the CR group was subdivided into CR1 and CR2, rats sacrificed within the first 24-h after feeding and within the following 24-h, respectively. Consequently, when sacrificed in the CR1 phase, the CR group consumed the similar amount of food as each AL group before sacrifice.

The body weights and food intake of rats in each group during the lifespan study are illustrated in Figure 31.1.

It should be noted that both the body weight and food intake of (—/—)-CR rats were similar to those of (tg/—)-AL rats for a considerable period of the experiment. Thus, comparative studies using (tg/—)-AL and (—/—)-CR rats could give insight into the role of the GH-IGF-1 axis in CR.

Pulsatile secretion of GH has yet to be analyzed in all rat groups; however, an immunohistochemical study revealed that the number of GH-positive cells was markedly reduced in (tg/tg) rats compared with (—/—) rats (Shimokawa et al., 2002). Plasma IGF-1 concentrations at 6 months of age are provided in Table 31.1. The IGF-1 concentration decreased by 43% in (tg/—)-AL rats and by 74% in (tg/tg)-AL rats compared with (—/—)-AL rats. CR further decreased the IGF-1 concentration in each rat group; values in the CR2 phase were slightly lower than in the CR1 phase. It should be noted that the IGF-1 level was slightly lower in (tg/—)-AL rats than (—/—)-CR rats.

Lifespan and Pathology

The survival curves of (—/—), (tg/—), and (tg/tg) rats fed ad libitum and the effect of CR on (—/—) and (tg/—) rats were published previously (Shimokawa et al., 2002; Shimokawa et al., 2003). At the 25th percentile survival point, the lifespan was increased by 10% in (tg/—)-AL rats compared with (—/—)-AL rats (Table 31.2). In contrast, the lifespan of (tg/tg)-AL rats was diminished by 9%. CR increased the lifespans of (—/—), (tg/—), and (tg/tg) rats by 11, 10, and 19%, respectively.

Postmortem examinations were performed to determine the prevalence of selected diseases and analyze probable causes of death. Prevalence of pituitary adenoma did not differ between (—/—)-AL and (tg/—)-AL rats, but was significantly decreased in (tg/tg)-AL rats (Table 31.3). CR reduced the prevalence of pituitary adenoma compared to the AL group in (tg/—) rats, while statistically insignificant in (—/—) and (tg/tg) rats. Prevalence of chronic nephropathy was also significantly reduced in (tg/—) and (tg/tg) rats, and similarly, CR also decreased the prevalence. Although the number of rats

Figure 31.1. (A) Mean body weights of each rat group in the longevity study; n = 30 for each group at the start of the study, except the (tg/tg)-CR group (n = 10). Data are not depicted when the number of rats was below five, because of spontaneous death. (B) Mean food intake (g/rat/2 days) of the rat groups in the longevity study (n = 10, respectively). Data are not depicted when the number of rats was below five.

Figure 31.1. (A) Mean body weights of each rat group in the longevity study; n = 30 for each group at the start of the study, except the (tg/tg)-CR group (n = 10). Data are not depicted when the number of rats was below five, because of spontaneous death. (B) Mean food intake (g/rat/2 days) of the rat groups in the longevity study (n = 10, respectively). Data are not depicted when the number of rats was below five.

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