The somatotropic axis and longevity in mice.

The somatotropic signaling pathway has been implicated in aging and longevity studies in mice and other species. The physiology and lifespans of a variety of mutant mice, both spontaneous and genetically engineered, have contributed to our current understanding of the role of growth hormone and insulin-like growth factor I on aging-related processes. Several other mice discovered to live longer than their wild-type control counterparts also exhibit differences in growth factor levels; however, the complex nature of the phenotypic changes in these animals may also impact lifespan. The somatotropic axis impacts several pathways that dictate insulin sensitivity, nutrient sensing, mitochondrial function, and stress resistance as well as others that are thought to be involved in lifespan regulation.

Longevity in mice: is stress resistance a common factor?

A positive relationship between stress resistance and longevity has been reported in a multitude of studies in organisms ranging from yeast to mice. Several mouse lines have been discovered or developed that exhibit extended longevities when compared with normal, wild-type mice of the same genetic background. These long-living lines include the Ames dwarf, Snell dwarf, growth hormone receptor knockout (Laron dwarf), IGF-1 receptor heterozygote, Little, alpha-MUPA knockout, p66(shc) knockout, FIRKO, mClk-1 heterozygote, thioredoxin transgenic, and most recently the Klotho transgenic mouse. These mice are described in terms of the reported extended lifespans and studies involving resistance to stress. In addition, caloric restriction (CR) and stress resistance are briefly addressed for comparison with genetically altered mice. Although many of the long-living mice have GH/IGF-1/insulin signaling-related alterations and enhanced stress resistance, there are some that exhibit life extension without an obvious link to this hormone pathway. Resistance to oxidative stress is by far the most common system studied in long-living mice, but there is evidence of enhancement of resistance in other systems as well. The differences in stress resistance between long-living mutant and normal mice result from complex interrelationships among pathways that appear to coordinate signals of growth and metabolism, and subsequently result in differences in lifespan.

Glutathione metabolism in long-living Ames dwarf mice.

Ames dwarf mice live significantly longer than their wild type siblings and exhibit elevated antioxidative defenses and reduced oxidative damage. This study was conducted to determine the levels of components of glutathione (GSH) synthesis, degradation and utilization in dwarf and wild type mice. Glutamate-cysteine ligase protein levels were significantly elevated in dwarf liver at 3 and 24 months of age and muscle tissue at all ages examined. In kidney, activity of gamma-glutamyltranspeptidase (GGT) was decreased 42, 30 and 33% in 3, 12 and 24-month-old dwarf mice compared to wild type mice (P<0.0001). In contrast, GSH-S-transferase (GST) activity was markedly elevated (85, 113 and 53%) in kidneys of 3, 12 and 24-month-old dwarf mice (P<0.0001). GGT activity was higher in hearts of young dwarf and wild type mice while GST activity tended to be greater in dwarf mice. Similar to liver and kidney, brain GGT activity was also lower in dwarf mice (P<0.0001). Results of these experiments coupled with previous data provide a mechanism to partially explain the enhanced resistance to oxidative insult and conceivably, the extended longevity of dwarf mice.

Differential effects of light/dark recombinant human prolactin administration on the submaxillary lymph nodes and spleen activity of adult male mice.

OBJECTIVES: Day/night variations in cellularity, percentage of CD4+, CD8+ and double-positive (CD4+-CD8+) lymphocytes, lipopolysaccharide (LPS)- and concanavalin A (Con A)-induced lymphocyte proliferation and natural killer (NK) activity, and the effect of timed administration of recombinant human prolactin (h-PRL) on the above-mentioned parameters were investigated in the submaxillary lymph nodes and spleen of adult male mice. RESULTS: In controls, the percentage of CD4+, double-positive lymphocytes, LPS- or Con A-induced blastogenic proliferation and NK activity in the spleen differ during the dark phase as compared to the light phase. When administered during the dark period, h-PRL induced immunosuppresion in the percentage of CD4+, double-positive (CD4+-CD8+) lymphocytes. Con A- and LPS-induced lymphocyte proliferation and NK activity as compared to untreated controls. When h-PRL was administered during the light period, the cellularity increased, and h-PRL was immunosuppressive in Con A- and LPS-induced lymphcoyte proliferation and NK activity as compared to controls. Moreover, in control submaxillary lymph nodes the cellularity, percentage of CD8+, double-positive lymphocytes, blastogenic proliferation in the presence of Con A and LPS and NK activity differ when comparing the dark with the light phase. When administered during the dark period h-PRL induced immunosuppression in the percentage of double-positive (CD4+-CD8+) lymphocytes, Con A- and LPS-induced lymphocyte proliferation as compared to controls. When h-PRL is administered during the light period, no effects were observed. CONCLUSIONS: These results indicate the existence of differential day/night variations in the cellular immune response depending upon the lymphoid organ considered. Because of the administration of h-PRL a differential modulation of this circadian variation was also observed.

Cardiac contractile function is enhanced in isolated ventricular myocytes from growth hormone transgenic mice.

Growth hormone (GH) plays a key role in cardiac growth and function. However, excessive levels of GH often result in cardiac dysfunction, which is the major cause of death in acromegalic patients. Transgenic mice with GH over-expression serve as useful models for acromegaly and exhibit impaired cardiac functions using echocardiography, similar to those of human acromegaly. However, the mechanism underscoring the impaired ventricular function has not been well defined. This study was designed to evaluate the cardiac excitation-contraction coupling in GH over-expressing transgenic mice at the single ventricular myocyte level. Myocytes were isolated from GH and age-matched wild-type mouse hearts. Mechanical properties were evaluated using an IonOptix MyoCam system. The contractile properties analyzed included peak shortening (PS), time-to-peak shortening (TPS) and time-to-90% relengthening (TR(90)), and maximal velocities of shortening/relengthening (+/-dL/dt). Intracellular Ca2+ properties were evaluated by fura-2. GH transgenic mice exhibited significantly increased body weights and enlarged heart and myocyte size. Myocytes from GH transgenic mice displayed significantly enhanced PS and+/-dL/dt associated with similar TPS and TR(90) compared with the wild-type littermates. Myocytes from GH transgenic mice displayed a similar resting intracellular Ca2+ level and Ca2+ removal rate but exhibited an elevated peak intracellular Ca2+ level compared with the wild-type group. Myocytes from both groups were equally responsive to increases in extracellular Ca2+ concentration and stimulating frequency. These results suggest that GH over-expression is associated with enhanced contractile function in isolated myocytes and that the impaired cardiac function observed in whole hearts may not be due to defects at the myocyte level.

Effects of overexpression of growth hormone on T cell activity in transgenic mice.

Growth hormone plays a key role in the maturation and maintenance of the immune response, however, the effects of chronic high circulating concentrations of the hormone on the immune system is poorly understood. Transgenic mice overexpressing bovine growth hormone (b-GH) gene, fused to the rat phosphoenolpyruvate carboxykinase promoter (PEPCK), with very high plasma concentration of heterologous b-GH and their littermate normal siblings were used. Spleen cellularity, percentages of total T lymphocytes, CD4+ and CD8+ cells, ratio of T cell subpopulations, mitogen-induced lymphocyte proliferation and natural killer (NK) cell activity were examined in male transgenic mice and normal littermate mice at 2 and 6 months of age. The number of splenic lymphocytes was greater in transgenic mice than in matched normal littermates at both ages. The NK cell activity was lower in transgenic mice than in the matched normal littermates at both ages, with the lowest values found in older mice. The b-GH transgenic mice had lower percentages of T cells at both ages, however, in young transgenic mice, the percentage of CD4+ cells was reduced while percentage of CD8+ cells was increased in comparison to normal controls. Both basal and mitogen-induced proliferation capacity of splenocytes were reduced in PEPCK-b-GH-25 mice as compared to normal littermates of both ages. Proliferative indexes in response to concanavalin A and phytohemagglutinin were markedly decreased in 6 month old PEPCK-b-GH-25 mice as compared to littermate controls or younger mice. These results indicate that overexpression of b-GH in mice is associated with decreased T cell function and that these abnormalities are age-dependent.

Prolonged longevity of hypopituitary dwarf mice.

In two types of mutant dwarf mice, congenital deficiencies in pituitary function are associated with remarkably increased life expectancy. In this review, we will describe the key phenotypic characteristics of these animals, the evidence that they exhibit delayed aging, and the mechanisms that are suspected to account for their prolonged longevity.

Mitochondrial oxidant generation and oxidative damage in Ames dwarf and GH transgenic mice.

Aging is associated with an accumulation of oxidative damage to proteins, lipids and DNA. Cellular mechanisms designed to prevent oxidative damage decline with aging and in diseases associated with aging. A long-lived mouse, the Ames dwarf, exhibits growth hormone deficiency and heightened antioxidative defenses. In contrast, animals that over express GH have suppressed antioxidative capacity and live half as long as wild type mice. In this study, we examined the generation of H2O2 from liver mitochondria of Ames dwarf and wild type mice and determined the level of oxidative damage to proteins, lipids and DNA in various tissues of these animals. Dwarf liver mitochondria (24 months) produced less H2O2 than normal liver in the presence of succinate (p<0.03) and ADP (p<0.003). Levels of oxidative DNA damage (8ÕHdG) were variable and dependent on tissue and age in dwarf and normal mice. Forty-seven percent fewer protein carbonyls were detected in 24-month old dwarf liver tissue compared to controls (p<0.04). Forty percent more (p<0.04) protein carbonyls were detected in liver tissue (3-month old) of GH transgenic mice compared to wild types while 12 month old brain tissue had 53% more protein carbonyls compared to controls (p<0.005). Levels of liver malonaldehyde (lipid peroxidation) were not different at 3 and 12 months of age but were greater in Ames dwarf mice at 24 months compared to normal mice. Previous studies indicate a strong negative correlation between plasma GH levels and antioxidative defense. Taken together, these studies show that altered GH-signaling may contribute to differences in the generation of reactive oxygen species, the ability to counter oxidative stress and life span.

Catalase expression in delayed and premature aging mouse models.

The physiological decline that occurs with aging is thought to result, in part, from accumulation of oxidative damage produced by reactive oxygen species (ROS) generated during normal metabolism. Two genetic mouse models of aging, the Ames dwarf and growth hormone (GH) transgenic, suggest that hormone levels may play a role in antioxidative defense and aging. To explore this possibility, catalase (CAT), an enzyme involved in elimination of ROS, was evaluated in long-lived dwarf and short-lived transgenic mice. Catalase activity and/or protein was significantly elevated in livers from dwarf mice at 3, 6, 13-15, and 24 months of age when compared to age-matched wild type mice. In contrast, a 50 and 38% reduction (P<0.05) in CAT protein was observed in 3 and 10 to 12 month old GH transgenics respectively, when compared to wild type mice. Kidneys from old dwarf mice exhibited significantly increased CAT activity (22%), protein (16%) and mRNA expression (59%) compared to wild type mice. Conversely, kidneys from GH transgenic mice showed reductions in CAT activity. The results of this study suggest that hormonal status modulates antioxidative mechanisms and that CAT is important in overall defense capacity with respect to lifespan in both decelerated (dwarf) and accelerated (transgenic) mammalian models of aging.

Growth hormone and aging.

The potential usefulness of growth hormone (GH) as an anti-aging therapy is of considerable current interest. Secretion of GH normally declines during aging and administration of GH can reverse age-related changes in body composition. However, mutant dwarf mice with congenital GH deficiency and GH resistant GH-R-KO mice live much longer than their normal siblings, while a pathological elevation of GH levels reduces life expectancy in both mice and men. We propose that the actions of GH on growth, development, and adult body size may serve as important determinants of aging and life span, while the age-related decline in GH levels contributes to some of the symptoms of aging.

Immune function in transgenic mice overexpressing growth hormone (GH) releasing hormone, GH or GH antagonist.

Effects of life-long exposure to high levels of homologous or heterologous growth hormone (GH) and effects of GH resistance on selected parameters of immune function were studied in adult male transgenic mice overexpressing GH releasing hormone (GHRH), bovine (b) GH or an antagonistic bGH analog. In metallothionein I (MT)-bGH transgenic mice with high peripheral levels of bovine GH, there were significant increases in the absolute weight of the thymus and the spleen and in the mitogenic responses of splenocytes to concanavalin A (ConA), lipopolysaccharide (LPS) and phytohemagglutinin (PHA), as compared to age-matched normal animals. There were no significant differences between MT-bGH transgenic and normal mice in splenocyte viability or in delayed-type hypersensitivity measured by the allergic contact dermatitis response to oxazolone. Similar results, including significant stimulation of splenocyte responses to ConA, LPS, and PHA, were obtained in MT-hGHRH transgenic mice in which overexpression of GHRH leads to striking pituitary enlargement and massive elevation of peripheral levels of homologous (mouse) GH. In MT-bGH-antagonist transgenic mice in which overexpression of an antagonistic bGH analog interferes with the actions of endogenous GH, spleen weight was reduced but proliferative responses of splenocytes to ConA, LPS, and PHA were not affected. It is concluded that overexpression of heterologous or homologous GH in transgenic mice can lead to significant stimulation of some parameters of immune function, whereas antagonism of GH action by expression of an antagonistic GH analog does not affect splenocyte responses to mitogens.

Antioxidative mechanisms and plasma growth hormone levels: potential relationship in the aging process.

Factors affecting longevity are complex and poorly understood. We have recently found that Ames dwarf mice (df/df), which are deficient in growth hormone (GH), prolactin, and thyroid-stimulating hormone, live significantly longer than their normal siblings whereas transgenic mice that overexpress GH exhibit reduced life-spans and various indices of premature aging. The production of reactive oxygen species increases with aging and is associated with DNA damage to the tissues. However, several cellular oxygen scavenging/detoxifying systems exist that improve the antioxidative defense capacity of cells. We evaluated the activity of enzymes involved in this defense system in liver, kidney, and heart tissue from dwarf, phosphoenol-pyruvate carboxykinase-bovine GH transgenic, and corresponding groups of normal mice. Liver glutathione and ascorbate levels were lower (p < 0.0025) in dwarf animals compared to normal and GH transgenic mice. By contrast, the level of catalase activity, which detoxifies hydrogen peroxide, in dwarf liver and kidney was significantly higher when compared to the other groups. Animals deficient in GH (dwarf) live longer and exhibit enzyme activities and levels that may combat oxidative stress more efficiently than normal mice and those overexpressing GH.

Does growth hormone prevent or accelerate aging?

It is very well documented that plasma growth hormone (GH) levels decline with age in the human and in experimental animals, and there is considerable evidence that age-related changes in body composition may be caused by reduced function of the GH-IGF-I system. However, excessive GH levels are associated with reduced life expectancy in acromegalic patients and with symptoms of accelerated aging in GH transgenic mice. Hereditary dwarf mice deficient in GH, prolactin, and TSH live much longer than their normal siblings. Possible mechanisms of delayed aging in dwarf mice include lower core body temperature and reduced oxidative processes. It is suggested that the controversies concerning the apparent potential of GH to both prevent and accelerate aging may be reconciled by interpreting the results in light of the negative relationship between body size and life span within a species.

Developmental aspects of prolactin receptor gene expression in fetal and neonatal mice.

The prolactin receptor (PRL-R), a member of the hematopoietin cytokine receptor superfamily, is widely distributed among mammalian tissues. To understand better the potential sites of action and onset of potential PRL responsiveness, the developmental distribution pattern of PRL-R mRNA expression in fetal and neonatal mice was examined. Fetal mouse tissues were collected at distinct stages from timed pregnancies. Following extraction of total RNA, onset of gene expression was evaluated via reverse transcription-polymerase chain reaction (RT-PCR) and Southern hybridization was employed for verification. Expression of PRL-R mRNA was first observed on day 14 in the liver and cranium and on day 15 in the kidney, lung and thymus gland. Pituitary and adrenal glands were positive for PRL-R at day 18 of gestation through to day 1 of postnatal life. Neither whole fetuses prior to day 14 (days 10-13) of gestation nor skin and bladder tissues from 2-day-old mice generated detectable RT-PCR signals for PRL-R. The presence of PRL-R mRNA in fetal thymus and spleen tissues suggests a possible role for PRL in the development of the immune system. Prolactin may act directly on the pituitary to influence its own secretion and/or that of other pituitary-derived factors, as evidenced by the presence of PRL-R mRNA in the pituitary glands of fetal and 1-day-old mice. These data are the first to show the presence of PRL-R gene expression in various organ systems in fetal mice and suggest that PRL is among several factors necessary to coordinate developmental activities.

Age-related effects of ectopic pituitary transplants on the activation of Ames dwarf mouse lymphocytes in vitro.

Prolactin (PRL), one of the anterior pituitary hormones, has been implicated in the development and maintenance of immune system function. The following experiments were conducted to evaluate age-related effects of PRL on immune system activity in a PRL-deficient mouse model, the Ames dwarf. Two- and ten-month-old dwarf and phenotypically normal male mice were used. Six dwarf mice from each age group received a surgically implanted pituitary (under the kidney capsule) from a normal donor female mouse. Six different dwarfs were similarly operated but had no pituitary graft inserted and will be referred to as sham-operated dwarfs. Three weeks following surgery, spleens were removed from the dwarfs and age-matched normal mice and splenocytes isolated. The two age groups will subsequently be referred to as "3-month-old" and "11-month-old", respectively, The splenocytes were used in mitogen-induced (concanavalin A (Con A); phytohemagglutinin (PHA); lipopolysaccharide (LPS)) proliferation assays, and histopaque isolated lymphocytes were used for T cell surface marker determination. Pituitary grafting increased body weights of dwarf mice although the weights were less than control mice independent of age. A similar pattern was observed for total number of splenocytes per spleen. In young animals, the relative number of splenocytes per gram of body weight increased in pituitary grafted dwarfs reaching values of control mice, whereas much smaller differences were observed in older animals. The relative percentage of CD4+ cells was reduced (P<0.01) in 3-month-old pituitary-grafted mice, compared with sham-operated dwarf mice, while no differences were observed between sham- operated dwarf and normal mice. Pituitary grafting did not affect the numbers of CD8+ cells. In 11-month-old animals, the relative percentages of CD4+ and CD8+ cells were greater (P<0.05) in sham-operated dwarf than in normal mice but not affected by pituitary grafting. At 3 months of age, proliferation of splenocytes in response to Con A was increased (P<0.05) in sham-operated dwarfs and reduced (P<0.05) in pituitary-grafted dwarfs compared with normal mice. In contrast, PHA stimulation of splenocytes was decreased (P<0.05) in sham-operated compared with normal, and was still lower in mice receiving an ectopic pituitary. Substantially different responses were obtained in 11-month-old animals. In response to Con A, splenocytes from sham-operated dwarfs exhibited a reduced (P<0.05) proliferative capacity as compared with normals, and proliferation was increased in pituitary-grafted as compared with sham-operated dwarfs. In the presence of PHA, the proliferative capacity of splenocytes was greater (P<0.05) in sham-operated dwarfs as compared to normals and pituitary grafting normalized this parameter. These results demonstrate differential effects of PRL deficiency (sham-operated dwarfs versus normal) and of PRL replacement (pituitary-grafted versus sham-operated) in young as compared with old dwarf mice on immune system activation.

Assessment of the primary adrenal cortical and pancreatic hormone basal levels in relation to plasma glucose and age in the unstressed Ames dwarf mouse.

Peripheral glucose concentrations in mammals are maintained within very narrow limits to provide a continuous, uninterrupted supply of this nutrient to tissues. Numerous factors have been shown to influence and/or regulate glucose levels. One such influence is growth hormone (GH) produced by the pituitary somatotrophs. Several animal models of hyposomatotropism are available in which GH secretion or actions are suppressed due to genetic abnormalities. One such model, the Ames dwarf mouse (df/df), has arisen from an autosomal recessive mutation in which GH-, prolactin- (PRL), and thyroid-stimulating hormone (TSH)-producing cell types of the anterior pituitary fail to develop. The current investigation examined the effects of GH deficiency on glucose, insulin, and corticosterone levels using male and female df/df mice and their normal (Df/-) littermates. Additionally, old and young females of both genotypes were used to determine whether aging and GH deficiency interact to influence insulin, corticosterone or glucose levels in these animals. Plasma samples collected from unstressed animals (normal, df/df; young [5 months], old [17-19 months]; male, female) were used. Glucose levels were lower (P < 0.05) in df/df than in Df/- mice regardless of sex and age. A sex difference in Df/- animals was evident--young and old females had significantly lower levels of glucose when compared with young Df/- males. Plasma insulin was elevated (P < 0.05) in old df/df females compared with young df/df and Df/- females. Young Df/- males had the highest insulin levels compared with all genotype and age groups. This observation paralleled results from glucose measurements. Corticosterone levels were highest in young Df/- females and lowest in young Df/- males, with df/df animals falling between these values. Plasma corticosterone levels in old Df/- females did not differ from the values measured in dwarfs. The present findings indicate that glucose and factors affecting glucose levels are altered in the df/df mouse. These results provide new insights into the roles GH may play in glucose metabolism and perhaps also in adiposity which is a common characteristic of Df/- aged females from this line of mice.

Functioning of the porcine pituitary-adrenocortical axis during neonatal development.

A study was conducted with neonatal boars to measure age-related changes in functioning of the pituitary-adrenocortical axis. Pigs were randomly assigned to control (n = 7-10/age) or treated (1-min restraint, n = 9-11/age) groups to be sampled at either 12, 19, or 26 days of age. Blood samples were taken via catheter 10 min before and 3, 10, and 20 min after restraint or at similar time intervals in controls. One day later, pigs were killed and adrenal glands obtained for ACTH receptor measurements. Basal plasma ACTH concentrations were greatest (p = 0.035) on day 12 when compared with later ages, but basal plasma cortisol concentrations were comparable at the three ages. Compared with controls, restraint elevated incremental plasma ACTH and cortisol responses at each age (p < 0.004). On day 12, maximal plasma ACTH (p = 0.0006) and incremental cortisol (p < 0.006) responses to restraint were greater than at later ages. Binding to adrenal ACTH receptors was greatest (p < 0.05) at day 13, which may help explain the apparently increased in vivo response of the adrenal gland to ACTH at this time. Restrained pigs had increased growth rates with increasing age (p = 0.016) whereas growth rates for control pigs did not differ with age. At day 27, 24 h after the 1-min restraint, body weights of restrained pigs exceeded those of control pigs (p = 0.045). At day 20, adrenal DNA and protein in pigs restrained 24 h previously were greater than in control pigs (p < 0.05). These data suggest age-related changes in functioning of the pituitary-adrenal axis in neonatal boars, and an absence of period during neonatal life when the porcine pituitary adrenocortical axis cannot respond to a stressor. The data also indicate both rapid and long-term responses of the adrenal to a very modest stressor and suggest an extreme sensitivity of neonatal pigs to environmental perturbations.

Lymphocyte proliferative responses in neonatal pigs with high or low plasma cortisol concentration after stress induced by restraint.

High plasma cortisol concentration is associated with perception of stress and reduced immune function in pigs. Neonatal pigs (12, 19, or 26 days old) were tested to determine maximal cortisol response to a mild restraint stressor. Pigs were fitted with indwelling jugular cannulas 4 days prior to restraint. One day before restraint, 10 ml of blood was removed for lymphocyte isolation and subsequent in vitro lymphocyte proliferation and interleukin 2 (IL-2) assays. On the day of restraint, blood samples were drawn 10 minutes before and 3, 10, and 20 minutes after holding each pig in a supine position for 1 minute. Plasma cortisol concentration was determined by use of radioimmunoassay. Pigs with maximal cortisol response greater than the mean value for that age group were classified in the high-responder (HIRES) group. Conversely, those with values lower than the mean maximal response were assigned to the low-responder (LORES) group. The HIRES pigs had larger relative adrenal gland weights and higher baseline and maximal cortisol responses, compared with LORES pigs (P = 0.0170, P = 0.0002, P = 0.0001, respectively). Mitogen-induced lymphocyte proliferative responses (to phytohemagglutinin, concanavalin A, and pokeweed mitogen) were 60% lower (P = 0.0037, P = 0.0432, P = 0.0103, respectively) in HIRES vs LORES pigs. In vitro IL-2 production did not differ between HIRES and LORES pigs.