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 6–9 months of age. Rats were fed ad libitum (AL) or

30% 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 the

presence of both common and separate mechanisms regulating the glucose–insulin system between CR and the reduced GH–IGF-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 GH–IGF-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 GH–IGF-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) 269–272

www.elsevier.com/locate/expgero

* Corresponding author. Tel.: þ81-95-849-7050; fax: þ81-95-849-7052.

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 manifested

phenotypes 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 22–25 8C; 12-h light/dark

cycle), 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

6–7 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 at 280 8C until assays for

insulin 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 8–9 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 or

CR), 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-f

ANOVA) after logarithmic transformation of data. Fisher’s

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 Fisher’s PSLD test after 1-f

ANOVA. **p , 0:05 versus 2 /2 (CR) by Fisher’s PSLD test after 1-f ANOVA.

H. Yamaza et al. / Experimental Gerontology 39 (2004) 269–272270

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 were

comparable with those in 2 /2 (CR) rats (Table 1). Plasma

concentrations 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 , 0:0001Þ: Blood glucose decreased in tg/2 rats (Tg

effect, p , 0:0001Þ; although the time-dependent alteration

was not affected (Tg £ Time, not significant). Blood

glucose was also reduced similarly by CR in both 2 /2

and tg/2 rats (Diet effect, p , 0:0001; Tg £ Diet, not

significant). The time-dependent changes in glucose con-

centrations were significantly affected by CR (Diet £ Time,

p , 0:05Þ: In AL rats, the blood glucose concentration

gradually 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; Diet

effect, 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 , 0:0006Þ: 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 , 0:0001Þ:

CR reduced blood glucose concentration in 2 /2 and tg/2

rats (Diet effect, p , 0:0001; Tg £ Diet, not significant;

Tg £ Diet £ Time, not significant), and Tg also reduced it in

AL and CR rats (Tg effect, p , 0:0001Þ:

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) 269–272 271

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 GH–IGF-1 axis, which were fed AL, manifested

similar findings. The property of the glucose–insulin 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 GH–IGF-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 GH–IGF-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.

References

Baudry, A., Leroux, L., Jackerott, M., Joshi, R.L., 2002. Genetic

manipulation of insulin signaling, action and secretion in mice. Insights

into glucose homeostasis and pathogenesis of type 2 diabetes. Eur. Mol.

Biol. Org. Rep. 3 (4), 323–328.

Bluher, M., Kahn, B.B., Kahn, C.R., 2003. Extended longevity in mice

lacking the insulin receptor in adipose tissue. Science (5606), 572–574.

Coschigano, K.T., Riders, M.E., Bellush, L.L., Kopchick, J.J., 1999.

Glucose Metabolism in Growth Hormone Receptor/Binding Protein

Gene Disrupted Mice, Eighty First Annual Meeteing of the Endocrine

Society, San Diego, CA, USA, p. 553.

Dominici, F.P., Hauck, S., Argentino, D.P., Bartke, A., Turyn, D., 2002.

Increased insulin sensitivity and upregulation of insulin receptor,

insulin receptor substrate (IRS)-1 and IRS-2 in liver of Ames dwarf

mice. J. Endocrinol. 173 (1), 81–94.

Escriva, F., Rodriguez, C., Cacho, J., Alvarez, C., Portha, B., Pascual-

Leone, A.M., 1992. Glucose utilization and insulin action in adult

rats submitted to prolonged food restriction. Am. J. Physiol. 263,

E1–7.(1 Pt 1).

Longo, V.D., Finch, C.E., 2003. Evolutionary medicine: from dwarf model

systems to healthy centenarians? Science 299 (5611), 1342–1346.

Matsumoto, K., Kakidani, H., Takahashi, A., Nakagata, N., Anzai, M.,

Matsuzaki, Y., et al., 1993. Growth retardation in rats whose growth

hormone gene expression was suppressed by antisense RNA transgene.

Mol. Reprod. Dev. 36 (1), 53–58.

Shimokawa, I., Higami, Y., Tsuchiya, T., Otani, H., Komatsu, T., Chiba, T.,

Yamaza, H., 2003. Lifespan extension by reduction of the growth

hormone–insulin-like growth factor-1 axis: relation to caloric restric-

tion. Faseb J. 8, 8.

Shimokawa, I., Higami, Y., Utsuyama, M., Tuchiya, T., Komatsu, T.,

Chiba, T., Yamaza, H., 2002. Life span extension by reduction in

growth hormone–insulin-like growth factor-1 axis in a transgenic rat

model. Am. J. Pathol. 160 (6), 2259–2265.

Strauss, E., 2001. Longevity. Growing old together. Science 292 (5514),

41–43.

H. Yamaza et al. / Experimental Gerontology 39 (2004) 269–272272