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* Department of Physiology, Southern Illinois University School of Medicine, Carbondale, Illinois 62901–6512;
Clinical Research Institute of Montreal, Montreal PQ H2W 1R7, Canada;
Edison Biotechnology Institute and Department of Biomedical Sciences, College of Osteopathic Medicine, Ohio University, Athens, Ohio 45701
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Key Words: growth hormone • thyroid hormones • insulin • thermoregulation • aging
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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).
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Another long-living mouse model that has deficiencies in the GH axis is the Snell dwarf mouse (9). The Snell dwarf has a recessive mutation at the Pit-1 locus that is unrelated to the mutation producing the Ames dwarf (10). This mutation, however, also leads to failure of differentiation of GH-, TSH-, and PRL-secreting cells. Thus, the Snell dwarf has an identical endocrine phenotype as the Ames dwarf (6–8, 10), and has also recently been shown to have a longer lifespan than its normal littermates (5). Although the mechanisms responsible for delayed aging in both of these dwarf mice are yet to be elucidated, these animals share a number of phenotypic characteristics with animals subjected to caloric restriction (CR). As such, the alterations common to both dwarf mice and CR can be suspected as being important for delaying the aging process.
Among the common characteristics of dwarf mice and CR animals are differences in GH and body size. In rats placed on a CR diet from weaning, GH hormone levels and body size are significantly lower than in ad libitum fed controls (11), although long-term CR preserves pulsatile GH release and thus eventually reverses GH suppression (12). In addition, CR significantly reduces levels of IGF-1 (12). In dwarf mice, GH is entirely absent and IGF-1 is consequently severely reduced (8). A second common characteristic among long-living mouse models is a reduction in body core temperature (Tco). Ames dwarf mice have consistently reduced Tco both during rest and following mild stress (14). Snell dwarf mice have reduced Tco and reduced metabolic rate (15, 16). CR animals also have reduced body temperature; however, they apparently have only transiently reduced metabolic rates (17). Differences in metabolism and Tco are consistent with the lack of TSH and the resulting hypothyroidism. Studies of CR animals indicate that thyroid hormones are reduced (18), and it has been similarly shown that induction of hypothyroidism increases the mean lifespan of normal rats (19). Yet another characteristic common among these animals is the regulation of insulin and glucose. Both glucose and insulin levels in CR animals have been shown to be consistently reduced (11, 20, 21). In the Ames dwarf, plasma glucose levels are lower than in normal mice, while plasma insulin is similar to normal mice in female dwarfs and reduced in male dwarfs (22).
Despite these similarities, the multiple endocrine deficits of the Ames and Snell dwarf mice make it difficult to interpret which characteristics may be a requisite for increased longevity and which are merely correlates. Recently, however, a new mouse model of GH deficiency was developed. These mice have targeted disruption (knockout [KO]) of the GH receptor/GH binding protein gene, thus eliminating GH signaling and greatly reducing IGF-1 levels, though GH levels are elevated (23). Homozygous (-/-) GHR-KO mice are small in size and have delayed puberty, although unlike the Ames dwarfs, both sexes are fertile (23, 24). Due to the very recent development of this line, its importance to the study of aging has only recently been reported. GHR-KO mice, like the Ames and Snell dwarfs, live significantly longer than either their heterozygous normal (+/-) or homozygous normal (+/+) littermates (25). Thus, with this single endocrine deficiency, the GHR-KO mouse may be a potentially useful and simpler model of increased longevity in mammals and should allow more specific identification of the endocrine and metabolic alterations that are important to extending lifespan.
The objectives of this study were to measure several metabolic and endocrine parameters in the GHR-KO mouse that are known to be affected by CR or altered in long-lived dwarf mice. These included measurement of Tco and thyroid hormones 3',3,5-L-triiodothyronine (T3) and thyroxine (T4). Nonfasted morning and afternoon glucose and morning insulin levels were also measured in these mice. Previous reports have shown that levels of glucose in males (26) and females (27) are reduced in GHR-KO mice following an overnight fast, but no data are available on glucose levels in these mice in the fed state or at different times of the day. In addition to these measurements, the level of corticosterone under nonstressed (morning and afternoon) and stressed conditions was determined. Corticosterone, which is an important component in the regulation of glucose levels, may also play a role in the aging process (11, 18, 20).
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Materials and Methods |
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Tco Measurement.
Tco was determined as previously described (14). Briefly, six female GHR-KO and normal littermates were anesthetized and a temperature-sensitive radio transmitter (XM-FH, Minimitter, Sun River, OR) was implanted into the abdominal cavity. Due to a limited number of receivers (three total), the measurements were conducted in four sets, each set on a separate day. For measurement of Tco, two mice (one GHR-KO and one normal) were transported in their home cage to the laboratory, each cage was placed on one of two telemetry readers (RA 1010, Minimitter), and the animals were allowed to acclimate for approximately 24 hr. For each of four trials (one GHR-KO and two normal mice, or the converse), Tco was recorded every 15 min for a 24-hr time period and was averaged per mouse for each hour.
Hormones/Glucose Levels.
Trunk blood was collected by decapitation or by cardiac puncture, following Isoflurane anesthesia, and was placed in tubes containing a 6% EDTA solution (50–100 µl depending on the size of the mouse), and was centrifuged at 5000 rpm at 4°C for 15 min. Plasma was collected and stored frozen until use.
For measurement of basal levels of glucose and basal corticosterone, mice of both sexes were separated two to three per cage on the evening prior to sacrifice. On the day of sacrifice, the mice were removed from the room where they were housed and were taken to an immediately adjacent separate area where they were sacrificed by decapitation. To minimize stress response, the time from initial cage disturbance to decapitation was less than 30 sec for each mouse. For this study, the animals were sacrificed starting either 1.5 hr after lights on (07:30 hr) or at 1.5 hr before lights off (16:30 hr) using eight GHR-KO and eight normal mice of each sex per time period. Plasma corticosterone was measured by radioimmunoassay (RIA) using a commercial kit (ICN Biomedicals, Costa Mesa, CA). Plasma glucose was measured using a colorimetric assay kit (Sigma, St. Louis, MO).
Corticosterone levels were also measured in blood plasma, collected at 09:00 hr, under stress conditions. For this study, 10 GHR-KO and 10 normal female mice were lightly anesthetized with Isoflurane, placed in a novel cage for 5 min, anesthetized again, and bled by cardiac puncture.
For measurement of thyroid hormones and insulin, female and male mice were sacrificed between 08:00 and 10:00 hr, using 10 GHR-KO and 10 normal mice of each sex. Plasma levels of T3 and T4 were measured in females only by commercial RIA kit (ICN Biomedicals). Insulin levels in females were measured by ICN RIA kit, while in males, insulin levels were measured by RIA kit from Diagnostic Products Corporation (DPC, Los Angeles, CA).
All RIA measurements were performed in duplicate within the same assay. Variation between duplicate samples of less than 5% was considered acceptable. The sensitivity for these assays were corticosterone, 25 ng/ml; T3, 25 ng/dl; T4, 1 µg/dl; insulin (ICN), 3 µIU/ml; and insulin (DPC), 4 µIU/ml.
Statistical Analysis.
Glucose and hormone measurement data were analyzed by ANOVA and Fisher's PLSD post hoc test for comparisons of more than two group means, and by Student's t test for comparisons of two means. For Tco, body temperature over each hour of the 24-hr recording period was averaged per mouse and was analyzed by Student's t test for comparison of GHR-KO versus normal mice. P < 0.05 was considered significant, and data are presented as the means ± SEM.
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Results |
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). Over this period, average Tco in GHR-KO mice was 0.4°C lower than normal mice (35.9° versus 36.3°C, respectively). However, these differences were statistically significant for only 6 hr during the 24-hr recording. These occurred during a single hour 4 hr prior to lights-off (14:00 hr), the initial hour after lights-off (18:00 hr), and during the 3 hr preceding lights on (03:00 to 06:00 hr).
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). Similarly, T4 levels in GHR-KO mice were also reduced by approximately one-third (P < 0.0005; Fig. 2b). However, the ratio of T3-to-T4 in GHR-KO mice did not differ between the two groups (P < 0.7974; Fig. 2c).
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). Glucose levels in normal mice tended to be higher in males than in females, while among GHR-KO mice these levels were similar between sexes. Male GHR-KO mice had reduced glucose levels when compared with normal males in both the morning and afternoon time measurements (P < 0.0003 and < 0.0001, respectively). Glucose levels in GHR-KO and normal male mice did not change significantly between the morning and afternoon determinations. In females, GHR-KO mice had normal levels of glucose in the morning (P < 0.1345). However, while glucose in normal females did not change significantly during the day, levels in GHR-KO females dropped significantly in the afternoon, such that this group had lower glucose than either afternoon normal females or morning GHR-KO females (P < 0.0263 and < 0.0009, respectively).
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). Normal females had apparently lower insulin levels than normal males (11.8 ± 2.95 µIU/ml). GHR-KO females had significantly lower insulin levels than normal females (3.964 ± 0.95 µIU/ml, P < 0.0213; Fig. 4b).
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). As expected, in each group, plasma corticosterone levels were elevated in the afternoon versus morning; however, these apparent differences were significant only in GHR-KO males (P < 0.0001). Comparisons between genotypes revealed that corticosterone levels in females were similar between GHR-KO and normal mice, while in males, plasma levels tended to be higher in GHR-KO mice. However, this was significant only in the afternoon (P < 0.0001). Following moderate stress of repeated anesthesia and exposure to a novel cage, GHR-KO and normal females responded with a substantial rise in corticosterone levels. Plasma corticosterone levels in stressed GHR-KO females were numerically higher than in stressed normal females, but this apparent difference was not statistically significant (Fig. 5b).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Results from measurement of Tco revealed that GHR-KO mice are only slightly hypothermic with respect to normal mice. While average Tco was significantly lower for six 1-hr intervals during the 24-hr recording period, during the remainder of the recording (i.e., 18 hr), Tco values measured in GHR-KO and normal mice were not significantly different. Presumably, reduced Tco is not among the key mechanisms of increased lifespan in GHR-KO mice. This contrasts with the findings in the Ames dwarf mouse in which Tco is consistently reduced (14). Lower body temperature resulting, perhaps, from reduced metabolic rate would potentially lead to the production of fewer free radicals, which are known to be related to the aging process (28). However, given the 30% to 50 % increase in lifespan seen in GHR-KO mice (26), the very moderate reductions in Tco reported here are quite unlikely to be responsible for such a dramatic effect.
Measurements of plasma T3 and T4 revealed that female GHR-KO mice are moderately hypothyroid when compared with their normal littermates. The consistent reductions in both thyroid hormones would suggest that there are no major alterations in thyroid function and that GHR-KO mice are only mildly hypothyroid. Thus, this long-lived model is again qualitatively similar to the Ames dwarf; however, quantitatively the phenotypic alteration is much milder in GHR-KO mice (29). These reductions are most likely secondary to GH resistance, since both GH and IGF-1 have facilliatory effects on the thyroid (8, 30). However, our results show that the presumably GH resistance-mediated reductions in thyroid hormone levels are mild when compared with those in the TSH and GH deficient Ames dwarf mouse.
The effects of GHR gene disruption on glucose metabolism and insulin levels measured in fed GHR-KO mice in the present study are not in complete agreement with reports in fasted GHR-KO mice (26, 27) or with results obtained in the Ames dwarf or CR animals (24, 13). Glucose levels in ad libitum-fed GHR-KO mice that were sacrificed in the morning were significantly reduced in males, but were only slightly and nonsignificantly lower in females when compared with normal mice. CR has consistently been shown to reduce glucose levels (11), and in the Ames dwarf, glucose levels are reduced in both sexes (22). However, the lack of reduced glucose levels in female GHR-KO mice in the morning may suggest a different diurnal pattern of glucose regulation in females than males, given the significant morning to afternoon reductions seen in female GHR-KO mice. Data on glucose levels in mice sacrificed in the afternoon, when the mice can reasonably be expected to have consumed little if any food during the immediately preceding light portion of the diurnal cycle, may be more comparable with the results obtained in fasted mice. Indeed, in the afternoon group, both male and female GHR-KO mice had significantly reduced glucose levels. Alternatively, other factors may have contributed to the differences between the results reported by Coschigano et al. (26) and those obtained in the present study, including the slightly different genetic background and use of both +/+ and +/- mice as normals, the assay method employed, or time and method of blood collection. In contrast, insulin levels were consistently reduced in GHR-KO mice. Reduced insulin and glucose levels have been shown in CR mice (31). However, in young Ames dwarf mice, insulin is not reduced in either sex (22). Comparisons of data obtained in GHR-KO, dwarf, and CR mice suggest that increased responsiveness of plasma glucose levels to insulin is a common feature of these three types of long-living animals, while reductions in plasma glucose and insulin are dependent on nutritional state and are somewhat less consistently observed.
Corticosterone levels in GHR-KO mice were generally higher than in normal mice, with GHR-KO males having significantly elevated levels over normal males in the afternoon only. Ames dwarf males, like GHR-KO mice, have elevated corticosterone levels, while female dwarf mice have lower corticosterone than normal mice (22). Corticosterone has been reported to have a negative effect on neurons in the hippocampus, a brain area important for learning and memory as an animal ages (32). However, no deficits in learning and memory were detected in GHR-KO or in Ames dwarf mice; in fact, these animals exhibit a delay in memory decline with age (33). Chronic mild elevation of corticosterone is consistently observed in CR rats and mice and is suspected of having a role in mediating the beneficial ``protective'' effects of reduced dietary intake in these animals (34).
Thus, several phenotypic features shared by GHR-KO, dwarf, and CR mice emerge as possible mechanisms of increased lifespan in these animals. However, the primary mechanism that may ultimately be responsible for long life in these mice is likely to be the deficiency of GH signaling. In support of the possible importance of GH and IGF-1 in the context of aging, small body size within a species correlates strongly with increased longevity in mice (35), dogs (36), and apparently also in the human (37). It has been further suggested that genes that accelerate growth and reproductive fitness early in life also accelerate aging and reduce lifespan (38). Given the report that GHR-KO mice have an increased lifespan over their normal siblings (26), our findings implicate an important role of the GH-IGF-1 axis in the determination of lifespan in mammals. Therefore, mutations of genes in this axis may reduce growth and reproductive fitness and concomitantly delay aging and prolong life. While the many possible mechanisms that directly or indirectly link GH to aging have not been fully elucidated, it has been shown that mitogenic signaling, oxidative stress, and longevity in mammals may be very closely related. In mice with targeted disruption of the p66shc stress response gene, growth factor signaling is blocked, while resistance to reactive oxygen species (ROS)-induced stress and longevity are significantly increased (39). These suggest that the lack of GH signaling in GHR-KO mice may be a causal factor in their delayed aging that is, perhaps, related to the decreased production or increased removal of free radicals. More work is needed in order to investigate this intriguing possibility. Furthermore, these data show that while some similarities exist between GH-resistant, GH-deficient, and CR animals, some of their characteristics vary widely, and some parameters suspected of involvement in the aging process may be unchanged. This would seem to suggest that there is no single mechanism for increased longevity in these different models, and that multiple factors, genetic or nutritional, may interact to delay aging. We are currently investigating the effects of caloric restriction in dwarf mice to characterize some of these interactions.
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Acknowledgments |
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Footnotes |
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1 To whom requests for reprints should be addressed at Southern Illinois University School of Medicine, Carbondale, IL 62901–6512. E-mail: shauck{at}siumed.edu 
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