Antioxidant enzymes, free-radical damage, and response to paraquat in liver and kidney of long-living growth hormone receptor/binding protein gene-disrupted mice.

Numerous studies have shown that the lifespan can be extended by caloric restriction or by altering the growth hormone (GH)-insulin-like growth factor 1 signaling pathway. Both of these manipulations produce physiological alterations, such as increased insulin sensitivity, and reduced glucose levels and body size. However, it is difficult to evaluate whether these are merely correlates of delayed aging or whether they have a direct causal effect on lifespan. One parameter that has been demonstrated to have causal, positive effects on longevity in invertebrates is improved antioxidant defenses. We measured activities of antioxidant enzymes Cu/Zn superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) and quantified free-radical damage by lipid peroxidation (LP) and protein oxidation (PO) measurements in liver and kidney tissues, and evaluated the response to paraquat-induced oxygen toxicity in the long-living GH receptor/binding protein gene knockout (GHR-KO) mouse. We found that in the kidney, SOD was lower and GPx was higher in GHR-KO mice, and LP was higher in female GHR-KO mice only. In the liver, female GHR-KO mice had lower GPx, while male GHR-KO mice had lower CAT and higher LP. GHR-KO males were also more susceptible to paraquat toxicity compared to females or normal males. We conclude that in long-living GHR-KO mice, GH-resistance does not confer longevity by improved free-radical scavenging in the liver and kidney, suggesting that greater free-radical defenses in other tissues, or altered glucose metabolism may have a more central role in extending the lifespan of these animals.

Reduced levels of thyroid hormones, insulin, and glucose, and lower body core temperature in the growth hormone receptor/binding protein knockout mouse.

The mechanisms that are responsible for the extension of lifespan in the mouse with targeted disruption (knockout [KO]) of the growth hormone (GH) receptor/binding protein (GHR-KO) are unknown. However, in the long-living Ames dwarf mouse, blood glucose and body core temperature (Tco) are consistently lower than in normal mice. In addition, insulin levels are reduced and corticosterone levels are elevated in male dwarfs. These functional alterations, similar to those seen in animals under caloric restriction, have not been proven to be causally related to the extension of lifespan, but they do provide some insight into what traits may be necessary for long life. Therefore, to investigate which of these parameters are similarly affected in two genetically unrelated, yet similarly long-living mouse models, we measured Tco, thyroid hormones (triiodothyronine [T3] and thyroxine [T4]), and insulin, in addition to morning and afternoon levels of glucose and corticosterone, in young adult male and/or female GHR-KO mice and their normal siblings. Tco in GHR-KO mice was numerically reduced throughout the 24-hr period; however, these differences were only significant 4 hr prior to lights-off (14:00 hr), immediately after lights-off (18:00 hr), and during the 3 hr preceding lights on (03:00 to 06:00 hr). GHR-KO mice had significantly reduced levels of T3 and T4, while the ratio of these hormones was similar to that in normal mice. Insulin levels in GHR-KO mice were lower than in normal mice; levels in male GHR-KO mice were below the detectable limits of the assay used. Glucose levels in GHR-KO mice (male and females) were lower than in normal mice in measurements taken in both morning and afternoon; however, these differences arose from consistent reductions in males, as morning glucose levels in GHR-KO females were similar to those of normal mice. Corticosterone levels measured in blood plasma collected under basal (nonstressed) conditions showed sex-related alterations. Basal corticosterone levels in female GHR-KO mice were similar to normal females, while those in male GHR-KO mice were higher than in normal males in the afternoon. Corticosterone levels in stressed GHR-KO females were similar to those measured in stressed normal females. These data show that the long-living GHR-KO mouse shares a reduction in glucose, insulin, thyroid hormones, and Tco with the Ames dwarf mouse. Reductions in these parameters may be important to the underlying mechanisms of delayed aging in these animals.

Free radical defenses in the liver and kidney of human growth hormone transgenic mice: possible mechanisms of early mortality.

The long-term effects of growth hormone (GH) administration are unknown. Although limited data on its short-term effects purport health benefits, numerous detrimental effects are the consequence of chronically elevated GH. We used spectrophotometric assay and Western blot to determine the effects of chronic GH excess on hepatic and renal antioxidant enzymes (AOEs) in young and middle-aged PEPCK (phosphoenolpyruvate carboxykinase) hGH (human GH) transgenic mice. In the liver, glutathione peroxidase (GPx) was reduced in transgenics of both age groups, catalase was reduced only in young transgenics, and Cu-Zn superoxide dismutase (SOD) was similar to normal mice, but declined with age. In all groups, hepatic AOE activity correlated significantly with AOE level. In the kidney, AOEs in young transgenics were similar to those of normal mice. However, middle-aged transgenics showed reduced renal SOD and GPx activities when compared with young transgenic or middle-aged normal mice. Similarly, renal SOD and GPx levels in middle-aged transgenics were reduced when compared with those of middle-aged normal mice. AOE activity in the kidney correlated significantly with AOE protein level among middle-aged animals only. These data suggest the following: ((1)) GH excess is associated with early declines in SOD and GPx in the kidney and reductions of hepatic GPx at all ages examined, perhaps increasing the risk of free radical-induced damage to these tissues; ((2)) in the liver of young animals and in the liver and kidney of middle-aged animals, AOE activity reflects the amount of enzyme protein; and ((3)) age-related reductions in GPx in transgenics may be related to the increased incidence of liver tumors and renal failure in these animals.

Effects of growth hormone on hypothalamic catalase and Cu/Zn superoxide dismutase.

Age-associated changes in hypothalamic catalase activity and level, and Cu/Zn superoxide dismutase (Cu/Zn SOD) activity were examined in Ames dwarf mice with growth hormone (GH) deficiency and prolonged lifespan, in PEPCK-hGH transgenic mice with overexpression of GH and reduced lifespan, and compared to values measured in normal controls. Hypothalami from young (3-4 months), middle-aged (9-10 months), and old (19-23 months) male mice were examined using spectrophotometric assay and Western blot. In dwarf mice, Cu/Zn SOD and catalase activities declined with age, and were higher than the corresponding normal values in young and middle-aged groups. Catalase levels also declined with age, but were similar to values in normal controls. In GH transgenic mice, age-associated decline of both catalase and Cu/Zn SOD occurred earlier than in normal animals. Catalase levels and activities in transgenic animals were similar to controls, whereas Cu/Zn SOD activity was higher in transgenics than in normal mice. The present results suggest that dwarf mice, during early life, have enhanced hypothalamic free radical defenses, which may contribute to their extended lifespan. However, from the present results in GH transgenic mice, it is impossible to conclude whether early decline of hypothalamic catalase and Cu/Zn SOD in these animals represents a correlate of accelerated aging, or contributes to their reduced lifespan.