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.

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.

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.

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.

Prolactin and growth hormone clearance in neonatal boars.

The pharmacokinetics of prolactin (PRL) and growth hormone (GH) are not known in neonatal pigs. In this study, six boars, ages 13 and 26 d, were used to determine metabolic clearance rate (MCR), half-life (t1/2), and volume of distribution (Vd) of immunoreactive porcine PRL (ipPRL) and immunoreactive porcine GH (ipGH). Blood samples were collected through indwelling jugular catheters at 0, 1, 2.5, 5, 10, 15, 30, 45, and 60 min after a 20 ng/g of BW (per hormone) cocktail of porcine PRL and GH. Analysis of the ipPRL disappearance curves indicated an apparent MCR of 13.79 +/- 2.45 and 23.79 +/- 4.10 mL/min for boars at 13 and 26 d of age, respectively (P = .0008). The t1/2 of ipPRL did not differ between ages (P = .9201). The apparent MCR of ipGH at 13 and 26 d were 10.60 +/- 2.09 and 27.30 +/- 4.55 mL/min, respectively, and this difference was still evident after adjustment for BW (P = .0465). The ipGH t1/2 decreased with age (P = .0232), 21.32 +/- 2.55 vs 13.57 +/- 3.35 min for 13 and 26 d, respectively. The apparent distribution volume for ipPRL was higher than that for ipGH at both ages (P = .0021). Within each age, the apparent MCR of ipGH and ipPRL did not differ significantly. These data represent the first clearance measures reported for ipPRL and ipGH in neonatal pigs and indicate that under the conditions of this study the apparent MCR of ipPRL and ipGH are similar but change significantly during early development.

Association between low birth weight and increased adrenocortical function in neonatal pigs.

This study examined differences in adrenocortical function between low- and high-birth-weight female neonatal pigs. Pigs born to unilaterally hysterectomized, ovariectomized sows were grouped by birth weight; "small" were < or = 1.2 kg and "large" were > 1.2 kg. Pigs were cross-fostered such that each sow had six to eight pigs that were either small or large. At 3 or 7 d of age a blood sample was obtained by venipuncture, pigs were killed, and adrenocortical cells were isolated. Adrenal weights (milligrams/kilogram BW) in small pigs were 46% greater (P = .001) than those in large pigs at both ages. Compared with those in large pigs, plasma cortisol concentrations were 70% greater (P = .006) in small pigs at 3 d and 199% greater at 7 d of age. Sensitivity of adrenocortical cell response to ACTH was enhanced at both ages in small pigs (P = .001). Corticosteroid binding globulin (CBG) binding capacity in small pigs was 75% greater (P = .03) than that in large pigs at 3 d and 26% greater at 7 d of age. Significant negative correlations existed between birth weight and relative adrenal weight, plasma cortisol, slopes of in vitro adrenocortical cell response curves to ACTH, and CBG binding capacity, irrespective of birth weight group classification. Such data indicate an association between adrenal function during early neonatal life and birth weight in female pigs. The current study does not indicate whether increased adrenal function causes or merely reflects conditions associated with low birth weight. However, we hypothesize that increased adrenal function is initiated prenatally and causes some instances of low birth weight.

In vivo neurotoxin administration alters immune responses in chickens (Gallus domesticus).

1. 6-Hydroxydopamine (6-OHDA) administered in ovo enhanced in primary immune response to sheep red blood cells (SRBC) in chicks 2. Splenic norepinephrine levels increased during the peak anti-SRBC response. 3. Cell-mediated immunity as measured by delayed-type hypersensitivity to phytohemagglutinin-P (PHA-P) was not affected by treatment with 6-OHDA.

Effect of fasting on plasma thyroid and adrenal hormone levels in turkey poults infected with Bordetella avium.

Plasma triiodothyronine (T3), thyroxine (T4), and corticosterone (CS) were measured in fasted and nonfasted control and Bordetella avium-infected poults. The stress of B. avium infection increased plasma CS, and fasting for 24 hours caused a further significant increase in CS levels. Plasma T3 was not affected by the infection, but fasting caused a significant reduction in both control and infected poults. Plasma T4 of fasted poults was increased in both control and infected groups, but infection attenuated the plasma T4 response. Total iodothyronines were increased in the control-fasted poults more than in infected-fasted poults, indicating a reduced responsiveness to stress by the thyroids of infected poults. Changes in plasma thyroid hormones and CS partially explain the decreased body weight gain and decreased body temperature after exposure to B. avium.

Catecholamine- and endotoxin-influenced cutaneous basophil hypersensitivity in chickens.

1. Catecholamines suppress cutaneous basophil hypersensitivity (CBH) to intradermal phytohemagglutinin (PHA-P) challenge in chickens. 2. E. coli 0127:B8 endotoxin, reserpine, and propranolol prevented a norepinephrine-induced suppression of CBH. 3. PHA-P induced CBH appeared to be mediated through a beta-adrenergic receptor mechanism associated with plasma corticosterone in chickens.

Tissue monoamine and plasma corticosterone concentrations following sympathetic alteration during acute endotoxicosis in chickens.

1. Splenic and heart ventricular NE levels and plasma corticosterone concentrations were increased following E. coli endotoxin administration in three-week-old chicks. 2. Chicks receiving either reserpine or propranolol injection before endotoxin exhibited increased NE in both tissues while only splenic 5-HT increased in birds receiving propranolol and endotoxin. 3. The effects of acute bacterial infection in chickens involves the peripheral nervous system neurotransmitters, NE and 5-HT, in the regulation of immune response.