A transgenic dwarf rat model as a tool for the study of calorie restriction and aging

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  • Short Communication

    A transgenic dwarf rat model as a tool for the study of calorie

    restriction and aging

    Haruyoshi Yamaza, Toshimitsu Komatsu, Takuya Chiba, Hiroaki Toyama, Kazuo To,Yoshikazu Higami, Isao Shimokawa*

    Department of Pathology and Gerontology, Nagasaki University Graduate School of Biomedical Sciences, 1-12-4 Sakamoto, Nagasaki City 852-8523, Japan

    Received 20 July 2003; received in revised form 29 October 2003; accepted 4 November 2003

    Abstract

    We have previously reported a long-lived transgenic dwarf rat model, in which the growth hormone (GH)-insulin like growth factor (IGF)-

    1 axis was selectively suppressed by overexpression of antisense GH transgene. Rats heterozygous for the transgene (tg/2) manifest

    phenotypes similar to those in calorie-restricted (CR) rats. To further characterize the transgenic rat in comparison with CR rats, the present

    study evaluated glucose and insulin tolerance in tg/2 and control Wistar (2 /2 ) rats at 69 months of age. Rats were fed ad libitum (AL) or30% CR from 6 weeks of age. In CR rats, glucose disposal after glucose load was facilitated without any significant surge of serum insulin,

    and insulin tolerance test also indicated increased insulin sensitivity. In transgenic rats, similar findings were observed after glucose and

    insulin load, and CR in tg/2 rats further facilitated glucose disposal during glucose and insulin tolerance tests. These findings suggest thepresence of both common and separate mechanisms regulating the glucoseinsulin system between CR and the reduced GHIGF-1 axis

    paradigms. The transgenic rat model is, therefore, a useful one for studies of CR and aging.

    q 2004 Elsevier Inc. All rights reserved.

    Keywords: Growth hormone; IGF-1; Calorie restriction; Glucose; Insulin; Transgenic rat

    1. Introduction

    Utilization of spontaneously mutated or genetically

    engineered rodent models that mimic physiological states

    induced by calorie restriction (CR) progresses our under-

    standing of the aging process and assists in developing anti-

    aging interventions in humans. We previously reported a

    long-lived transgenic dwarf rat model, in which the growth

    hormone (GH)-insulin like growth factor (IGF)-1 axis was

    selectively suppressed by overexpression of an antisense

    GH transgene (Shimokawa et al., 2002). These rats share

    some phenotypes with CR rats, including longer lifespan,

    some pathologies, reduced body size and food intake, and

    lower plasma levels of insulin, glucose, and IGF-1

    (Shimokawa et al., 2003).

    In lower organisms such as nematodes and fruit flies, in

    which insulin and IGF-1 systems are not clearly separated,

    functional mutations in insulin- or IGF-1-signaling expand

    lifespan (Strauss, 2001). In rodents, genetic mutations in this

    signaling also increase lifespan, although most direct

    manipulations of insulin signaling induce metabolic impair-

    ments such as diabetes and shorten lifespan (Baudry et al.,

    2002). Nonetheless, the glucose-insulin system appears

    important for regulation of aging and longevity in mammals;

    recent studies demonstrate that a reduced GHIGF-1 axis

    concomitantly modulates the glucose-insulin system, simi-

    lar to CR (Longo and Finch, 2003), and that a fat-specific

    knock out of the insulin receptor gene increases lifespan in

    mice (Bluher et al., 2003).

    Comparative studies using CR and rodent models with

    the reduced GHIGF-1 axis could facilitate our under-

    standing of the molecular mechanisms of aging and

    longevity. In this short communication, we described a

    transgenic dwarf rat with glucose and insulin tolerance and

    indicated the suitability of the model for future CR and

    aging studies.

    0531-5565/$ - see front matter q 2004 Elsevier Inc. All rights reserved.

    doi:10.1016/j.exger.2003.11.001

    Experimental Gerontology 39 (2004) 269272

    www.elsevier.com/locate/expgero

    * Corresponding author. Tel.: 81-95-849-7050; fax: 81-95-849-7052.E-mail address: shimo@net.nagasaki-u.ac.jp (I. Shimokawa).

  • 2. Materials and methods

    2.1. Animals

    The details of the rats and their husbandry in our

    laboratory are described elsewhere (Shimokawa et al., 2003,

    2002). The transgenic rats (Jcl: Wistar-TgN (ARGH-

    GEN)1Nts) were kindly provided by Nippon Institute for

    Biological Science (Oume City, Tokyo, Japan) and the

    present rat colony has been established in a barrier facility in

    the Laboratory Animal Center at Nagasaki University

    School of Medicine since 1997. The transgene consisted

    of four copies of the thyroid hormone response element, the

    rat GH promoter, and antisense cDNA sequences for rat GH

    (Matsumoto et al., 1993). Male rats heterozygous for the

    transgene (tg/2 ) were used, because those rats manifestedphenotypes similar to those in control non-transgenic Wistar

    (2 /2 ) rats subjected to CR (Shimokawa et al., 2003).Control male Wistar rats were purchased from Japan Clea,

    Inc. (Tokyo, Japan).

    At 4 weeks of age, weanling male rats were transferred to

    a barrier facility (temperature 2225 8C; 12-h light/darkcycle), kept separately under a specific pathogen-free

    condition, and fed ad libitum (AL) with Charles River-

    LPF diet (Oriental Yeast Co. Ltd. Tsukuba, Japan). At

    6 weeks of age, 30% CR was started by providing 140% of

    the mean food intake for 2 days in each group AL every

    other day. General data for each rat group at 6 months of age

    are presented as a reference (Table 1).

    All experiments reported here were performed in accord

    with provisions of the Ethics Review Committee for Animal

    Experimentation at Nagasaki University.

    2.2. Glucose tolerance test

    Glucose tolerance tests (GTT) were performed on rats at

    67 months of age. After a 15-h overnight fast, rats were

    injected intraperitoneally with D-glucose (1.0 g/kg body

    weight; 50% solution) and blood samples were withdrawn

    from tail veins at 0, 15, 30, 60, 90, and 120 min after glucose

    load without anesthesia using a 24-gauge needle. When

    collected a blood sample at each time point, a rat was placed

    into a body-sized-adjusted plastic box restrainer and blood

    was obtained in less than 60 s from the contact with the rat.

    In this experiment, blood samples were taken from each rat

    at only two time points because of the difficulty of repetitive

    samplings in the same animals. Group 1 rats were subjected

    to blood samplings at 0 and 60 min, while group 2 at 15 and

    90 min, and group 3 at 30 and 120 min. Blood glucose

    concentration was immediately measured with ACCU-

    CHEKw Active (Roche Diagnostics GmbH, Tokyo,

    Japan). Serum samples were also prepared by centrifugation

    of blood samples, and then stored at280 8C until assays forinsulin concentrations. Serum insulin levels were measured

    at 0, 15, 30, and 60 min with a rat insulin enzyme-

    immunoassay system (Amersham Pharmacia Biotech, Little

    Chalfont, UK).

    2.3. Insulin tolerance test

    The rats used for GTT were also subjected to insulin

    tolerance tests (ITT) at 89 months of age, and were fasted

    again for a 15-h before ITT. Blood samples were withdrawn

    from tail veins after intraperitoneal injection of human

    insulin (0.75 units/kg body weight; 1 unit/ml solution,

    Sigma Chemical Co., St Louis, MO) without anesthesia

    following the same procedure for GTT. Blood glucose

    concentration was also measured with ACCU-CHEKw

    Active (Roche Diagnostics GmbH).

    2.4. Statistics

    Data were presented as means ^ SD Blood glucose and

    serum insulin concentrations were analyzed for the main

    effect of transgene (Tg; 2 /2 or tg/2 ), diet (Diet; AL orCR), time (Time; 0, 15, 30, 60, 90, or 120 min), and their

    interaction (Tg Diet, Tg Time, Diet Time, Tg Diet Time) by three-factor analyses of variance (3-fANOVA) after logarithmic transformation of data. Fishers

    protected least significant difference (PLSD) test was also

    Table 1

    General data

    2 /2 tg/2

    AL CR AL CR

    Body weight (g) 481.4 ^ 23.9 (21) 336.9 ^ 23.3 (21) 308.7 ^ 14.3 (19) 209.1 ^ 14.8 (20)

    Food intake for 2 days (g) 45.3 ^ 2.7 (10) 31.7 31.7 ^ 2.4 (10)* 22.2

    IGF-1 (ng/ml) 1058.3 ^ 127.2 (12) 818.3 ^ 82.3 (12) 626.5 ^ 89.6 (5)** 345.6 ^ 39.8 (5)

    Glucose (mg/dl) 126.1 ^ 33.9 (5) 107.1 ^ 10.2 (8) 105.5 ^ 18.0 (5) 90.2 ^ 13.1 (8)

    Insulin (ng/ml) 101.8 ^ 48.8 (5) 16.0 ^ 9.3 (8) 21.6 ^ 17.7 (5) 23.5 ^ 26.0 (8)

    Values represent the mean ^ SD (the number of rats examined). All data was measured at 6 months of age. The data of IGF-1, glucose, and insulin are

    cited from the paper of Shimokawa et al (2003). Results of 2-f ANOVA on body weight are (1) body weight: Tg effect, p , 0:0001; CR effect, p , 0:0001;

    Tg CR, p , 0:0001; (2) IGF-1: Tg effect, p , 0:0001; CR effect, p , 0:0001; Tg CR, not significant (ns), (3) Glucose: Tg effect, p , 0:05; CR effect,p , 0:05; Tg CR, ns, (4) Insulin: Tg effect, p , 0:05; CR effect, p , 0:01; Tg CR, p , 0:05; *p , 0:0001 vs 2 /2 (AL) by Fishers PSLD test after 1-fANOVA. **p , 0:05 versus 2 /2 (CR) by Fishers PSLD test after 1-f ANOVA.

    H. Yamaza et al. / Experimental Gerontology 39 (2004) 269272270

  • performed as a post hoc test. One-factor ANOVA and the

    post hoc test were also carried out as needed for multiple

    comparisons. The level of significance was set at p , 0:05:

    3. Results

    3.1. General data

    The body weight and food intake in tg/2 (AL) rats werecomparable with those in 2 /2 (CR) rats (Table 1). Plasmaconcentrations of IGF-1, glucose, and insulin in the fed-state

    had been previously determined (Shimokawa et al., 2003).

    3.2. Glucose tolerance test

    As a whole, blood glucose concentration was increased

    to a peak value at 15 min after glucose load, and gradually

    returned to basal (0 min) level (Fig. 1(a); T effect,

    p , 0:0001: Blood glucose decreased in tg/2 rats (Tgeffect, p , 0:0001; although the time-dependent alterationwas not affected (Tg Time, not significant). Bloodglucose was also reduced similarly by CR in both 2 /2and tg/2 rats (Diet effect, p , 0:0001; Tg Diet, notsignificant). The time-dependent changes in glucose con-

    centrations were significantly affected by CR (Diet Time,p , 0:05: In AL rats, the blood glucose concentrationgradually decreased between 15 and 90 min; however, in

    CR rats, it quickly returned to basal level at 30 min.

    Serum insulin concentration during GTT was affected by

    CR, Tg, and Time (Fig. 1(b); Tg effect, p , 0:0001; Dieteffect, p , 0:0001; Time effect, p , 0:0001); however,there were also significant interactions between and among

    the factors (Tg Diet, p , 0:008; Diet Time, p , 0:007;Tg Diet Time, p , 0:0006: The concentration of

    insulin was transiently increased at 15 min in 2 /2 (AL)rats, and the level reduced precipitously to basal level at

    30 min. There was no similar surge of insulin in the other

    three groups of rats.

    3.3. Insulin tolerance test

    Blood glucose concentration decreased gradually

    between 15 and 90 min after insulin injection and stayed

    constant until 120 min (Fig. 2; Time effect, p , 0:0001:CR reduced blood glucose concentration in 2 /2 and tg/2rats (Diet effect, p , 0:0001; Tg Diet, not significant;Tg Diet Time, not significant), and Tg also reduced it inAL and CR rats (Tg effect, p , 0:0001:

    Fig. 1. (A) Blood glucose concentration during glucose tolerance testing. Data represent means ^ SD of four to seven rats. *p , 0:05 vs each correspondent

    group AL at 30 min. **p , 0:005 vs tg/2 (AL) and 2 /2 (AL), and p , 0:05 vs 2 /2 (CR) by multiple comparisons at 60 min. (B) Serum insulin

    concentration during glucose tolerance testing. Data represent means ^ SD of three to six rats. #p , 0:0001 vs the other three groups by multiple comparisons

    at 15 min. ##p , 0:05 vs 2 /2 (AL) by multiple comparisons at 30 min.

    Fig. 2. Blood glucose concentration during insulin tolerance testing. Data

    represent means ^ SD of five to six rats. *p , 0:05 vs each correspondent

    group AL, **p , 0:0001 vs tg/2 (AL), #p , 0:005 vs tg/2 (AL) and

    2 /2 (CR), and ##p , 0:05 vs 2 /2 (AL) by multiple comparisons at each

    time point.

    H. Yamaza et al. / Experimental Gerontology 39 (2004) 269272 271

  • 4. Discussion

    The present results were comparable to those in previous

    studies indicating that CR improves glucose tolerance and

    enhances insulin sensitivity in rodents (Escriva et al., 1992).

    Blood glucose concentration returned quickly to basal level

    without an insulin surge in response to exogenous glucose;

    and ITT also confirmed increased insulin sensitivity under

    CR conditions. Interestingly, transgenic rats with the

    reduced GHIGF-1 axis, which were fed AL, manifested

    similar findings. The property of the glucoseinsulin system

    in the transgenic rat was different from those in long-lived

    Ames dwarf and GHR-KO mice; as those mice models show

    glucose intolerance, although insulin sensitivity is increased

    (Coschigano et al., 1999; Dominici et al., 2002). In this

    respect, our transgenic rat model mimics the physiological

    state induced by CR more closely than the dwarf mice

    models.

    In the present model, CR further augmented glucose

    disposal without any significant change of serum insulin.

    These findings suggest not only that CR modulates glucose

    metabolism independently from the GHIGF-1 axis, but

    also that CR enhances insulinotropic or non-insulin

    dependent mechanisms for glucose disposal. Further

    analyses will be needed to elucidate differences between

    CR and the reduced GHIGF-1 axis in the mechanisms that

    underlie increased insulin sensitivity and glucose

    metabolism.

    Considering previously presented data on longevity and

    pathology, and together with the general data presented here

    in Table 1, we conclude that our transgenic dwarf rat is

    suitable for molecular analyses on the anti-aging effects of

    CR; as well as for assessing the relationship between

    longevity and insulin/IGF-1 signalings.

    Acknowledgements

    We thank Yutaka Araki and the staff in the laboratory

    animal center at Nagasaki University School of Medicine

    for their excellent technical support. We also thank Nippon

    Institute for Biological Science for providing the transgenic

    rat. This work was supported by the Research Grant for

    longevity Sciences (grants 11-C) from the Ministry of

    Health, Welfare, and Labor of Japan.

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