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A transgenic dwarf rat model as a tool for the study of calorie restriction and aging
Experimental Gerontology 39 (2004) 269–272 www.elsevier.com/locate/expgero
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 understanding of the aging process and assists in developing antiaging 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, * Corresponding author. Tel.: þ 81-95-849-7050; fax: þ81-95-849-7052. E-mail address: [email protected] (I. Shimokawa). 0531-5565/$ - see front matter q 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.exger.2003.11.001
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 impairments 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, similar 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 understanding 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.
270
H. Yamaza et al. / Experimental Gerontology 39 (2004) 269–272
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 (ARGHGEN)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 RiverLPF 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 ACCUCHEK w Active (Roche Diagnostics GmbH, Tokyo, Japan). Serum samples were also prepared by centrifugation of blood samples, and then stored at 2 80 8C until assays for insulin concentrations. Serum insulin levels were measured at 0, 15, 30, and 60 min with a rat insulin enzymeimmunoassay 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
Body weight (g) Food intake for 2 days (g) IGF-1 (ng/ml)‡ Glucose (mg/dl)‡ Insulin (ng/ml)‡
tg/2
AL
CR
AL
CR
481.4 ^ 23.9 (21) 45.3 ^ 2.7 (10) 1058.3 ^ 127.2 (12) 126.1 ^ 33.9 (5) 101.8 ^ 48.8 (5)
336.9 ^ 23.3 (21) 31.7 818.3 ^ 82.3 (12) 107.1 ^ 10.2 (8) 16.0 ^ 9.3 (8)
308.7 ^ 14.3 (19) 31.7 ^ 2.4 (10)* 626.5 ^ 89.6 (5)** 105.5 ^ 18.0 (5) 21.6 ^ 17.7 (5)
209.1 ^ 14.8 (20) 22.2 345.6 ^ 39.8 (5) 90.2 ^ 13.1 (8) 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) 269–272
271
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.
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:
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. Results
3.3. Insulin tolerance test
3.1. General data
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:
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 concentrations 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
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.
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H. Yamaza et al. / Experimental Gerontology 39 (2004) 269–272
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., PascualLeone, 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 restriction. 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.
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 understanding of the aging process and assists in developing antiaging 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, * Corresponding author. Tel.: þ 81-95-849-7050; fax: þ81-95-849-7052. E-mail address: [email protected] (I. Shimokawa). 0531-5565/$ - see front matter q 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.exger.2003.11.001
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 impairments 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, similar 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 understanding 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.
270
H. Yamaza et al. / Experimental Gerontology 39 (2004) 269–272
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 (ARGHGEN)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 RiverLPF 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 ACCUCHEK w Active (Roche Diagnostics GmbH, Tokyo, Japan). Serum samples were also prepared by centrifugation of blood samples, and then stored at 2 80 8C until assays for insulin concentrations. Serum insulin levels were measured at 0, 15, 30, and 60 min with a rat insulin enzymeimmunoassay 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
Body weight (g) Food intake for 2 days (g) IGF-1 (ng/ml)‡ Glucose (mg/dl)‡ Insulin (ng/ml)‡
tg/2
AL
CR
AL
CR
481.4 ^ 23.9 (21) 45.3 ^ 2.7 (10) 1058.3 ^ 127.2 (12) 126.1 ^ 33.9 (5) 101.8 ^ 48.8 (5)
336.9 ^ 23.3 (21) 31.7 818.3 ^ 82.3 (12) 107.1 ^ 10.2 (8) 16.0 ^ 9.3 (8)
308.7 ^ 14.3 (19) 31.7 ^ 2.4 (10)* 626.5 ^ 89.6 (5)** 105.5 ^ 18.0 (5) 21.6 ^ 17.7 (5)
209.1 ^ 14.8 (20) 22.2 345.6 ^ 39.8 (5) 90.2 ^ 13.1 (8) 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) 269–272
271
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.
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:
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. Results
3.3. Insulin tolerance test
3.1. General data
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:
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 concentrations 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
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.
272
H. Yamaza et al. / Experimental Gerontology 39 (2004) 269–272
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., PascualLeone, 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 restriction. 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.