Journal ofGerontology: BIOLOGICAL SCIENCES
Copyright 1999 by The Gerontological Society ofAmerica
1999,Vol.54A, No. 12,B521-B538
Pleiotropic Effects of Growth Hormone and Insulin-like
Growth Factor (IGF)-l on Biological Aging:
Inferences From Moderate Caloric-Restricted Animals
WilliamE. Sonntag, Colleen D. Lynch, WilliamT. Cefalu, RhondaL. Ingram,
SeanA. Bennett,Phillip L. Thornton, and Amir S. Khan
Department of Physiology and Pharmacology, WakeForestUniversity Schoolof Medicine, Winston-Salem, NorthCarolina.
Moderate caloric restriction (60% ofad libitumintake)is an imponantmodelto investigate potentialmechanisms ofbiologicalaging. This regimen has beenreported to decrease the numberofpathologies and increase lifespanin all species tested
to date. Although moderate caloric restriction inducesa widerangeof physiological changeswithinthe organism, adaptive
changeswithinthe endocrine systemare evidentand serveto maintainbloodlevels ofglucose. Thesealterations includean
increasein growth hormone secretory dynamicsand a declinein plasma levelsofIGF-l. Theseendocrinecompensatory
mechanismscan be inducedat any age,and wehaveproposedthatthesealterations mediatesomeofthe beneficialaspects
ofmoderatecaloricrestriction. Numerous studies indicatethat growth hormone and IGF-l decrease with age and that
administration ofthesehormonesameliorates the deterioration oftissuefunction evidentin agedad libitum-jedanimals,
suggesting that the absenceofthesehormonescontributes to thephenotypeofaging.Nevertheless, IGF-l is an importan:
risk factor in age-relatedpathologiesincluding lung, breast,and prostate cancer. From these studies, wepropose that
endocrinecompensatory mechanismsinducedby moderatecaloricrestriction (includingincreasedgrowthhormone and
decreased IGF-l) decrease the stimulusfor cellularreplication, resulting in a declinein pathologies and increased life span
observedin these animals. Thesefindings have important implicationsfor potential mechanisms ofmoderate caloric
restriction and suggestthat neuroendocrine compensatory mechanisms exerta key roleon the actionsofmoderatecaloric
restriction on life span.
SUBSTANTIAL volume of empirical and scientific evidence has accumulated, demonstrating that biologicalaging
is associated with functional deficitsat the cellular,tissue, organ,
and system levels (I). Several theories to explain these changes
as well as the increased risk of disease with age have been proposed. However, to date, no single explanation has adequately
accounted for the diversity of physiological changes associated
with age, and it is likely that the declinein tissue function and increased incidence of disease are the result of several interacting
factors. Current concepts of aging encompass free radical, somatic mutation, DNA repair, and cellular theories (2). Although
there are data to support each of these hypotheses, there is a
paucity of evidenceto suggest that the naturalprogressionof biologicalaging can beexplainedby these classicaltheories.
The neuroendocrine theory of aging (3) is an alternative hypothesis that was developed to account for physiological
changes associated with age. This concept evolved from studies
that indicatedthat (a) the endocrine system has an important role
in developmental processes; (b) hormone deficiency results in
deterioration of tissue function; and (c) hormones have an important trophic and integrative role in maintaining tissue function. The neuroendocrine system is composed of the hypothalamus and pituitary gland and is under the influence of
neurotransmitters and neuropeptides that regulatehypothalamicreleasing and inhibiting hormones secreted into the hypophysial
portal blood (4). The release of these hypothalamic hormones
influences the secretion of anterior pituitary hormones that subsequently regulate tissue function. The hypothalamus and pituitary have the capacity to detect humoral secretions from target
A
tissues and adjust hormone production to maintain an optimal
internal "milieu" appropriate for normal tissue function. It is
well established that the neuroendocrine system has a critical
role in regulating tissue growth throughout development and influences: (a) general metabolism of various tissues through the
release of growth hormone and thyroid-stimulating hormone;
(b) reproductive function through the release oflutenizing hormone, follicle-stimulating hormone, prolactin, and oxytocin;
and (c) plasma electrolytesand responses to stress through regulation of vasopressin and adrenocorticotropichormone. In addition, the hypothalamus has an important role in the integration
of parasympathetic and sympathetic activity and can thereby influence a wide variety of functions including heart rate, blood
pressure, vascular responses, and glucose metabolism, among
others. More recently, the hypothalamus has been implicated in
regulation of biological rhythms by its interactions with the
suprachiasmatic nucleus and other associated hypothalamic nuclei (5,6).The classification of hormones and their primary function noted above is an overly simplistic view of the neuroendocrine system, as critical interactions occur among hormones
that also contribute to the regulation of cellular and tissue function. Because many of the early events of aging include alterations in systems regulated by the neuroendocrine axis, it was
hypothesized that age-dependent alterations in the neuroendocrine system have the potentialto result in a progressiveseries
of events that contributeto the aging phenotype.
Although the etiology of neuroendocrine changes with age is
unknown, it has been proposed that cellular and molecular alterations in specific subpopulationsof neurons within the hypothaB521
B522
SONNTAG ETAL.
lamus and pituitary and/or supporting structures within the brain
are a contributing factor in the decrease in tissue function (7).
The cause of the specific perturbations may be related to loss of
neurons, genetic error, or the production of free radicals, which
lead to progressive aberrations in tissue function that facilitate
biological aging and the progression of disease. Thus, the neuroendocrine theory of aging is unique when compared to other
theories of aging in that neuroendocrine alterations are, in many
cases, not considered the primary causative factor of biological
aging but rather are considered to be mediators of aging initiated
by cellular changes in specific subpopulations of neurons or systems that closely interact with hypothalarnic neurons.
One of the few manipulations that have been reported to consistently influence the rate of biological aging is moderate
caloric restriction. Under this regimen, animals are fed a caloricrestricted diet (60% of ad libitum intake) utilizing a vitaminand mineral-supplemented diet from an early age (4 months in
our studies) throughout life and maintain a stable, albeit lower,
body weight than age-matched ad libitum controls. This regimen (generally termed moderate caloric restriction to contrast
with studies of acute starvation) increases both mean and maximal life span (8-12), reduces the appearance of pathological lesions 00,13,14), decreases susceptibility to chemical toxicity,
and increases DNA repair (15-19) and tissue protein synthetic
activity (20-22). Although most of the aforementioned studies
are well documented for rodent species, there are recent data
that find comparable effects of caloric restriction in nonhuman
primates (23-25). By comparing physiological adaptations in
the caloric-restricted animal to the ad libitum-fed animal over
the life span, this paradigm has been used to identify potential
biological mechanisms of aging that could then be pursued in
more rigorously controlled studies.
Our interest in pursuing the relationship between moderate
caloric restriction and neuroendocrine changes with age was
initially based on several lines of evidence: (a) with advancing
age, there is an increase in catabolic processes, most notably a
decline in cellular protein synthesis (26,27); (b) anabolic hormones such as growth hormone and insulin-like growth factor
(IGF-1) decrease with age (28,29), and restoration of these hormones increases cellular protein synthesis in a number of tissues (30,31); (c) moderate caloric restriction increases protein
synthesis in tissues, suggesting that catabolic events characteristic of aging are delayed or prevented by this regimen
(20-22,32); and (d) growth hormone and IGF-1 are critical for
the modulation of glucose homeostasis, and caloric restriction
would be expected to induce physiological changes in these
hormones to compensate for the reduction in caloric intake
(33). From preliminary studies, it was evident that IGF-1 decreased approximately 40% in response to moderate caloric restriction in young animals, but the long-term consequences of
dietary manipulation were unknown.
In this review, we present our results on the relationship between moderate caloric restriction and age-related changes in
the growth hormonelIGF-1 axis and the neuroendocrine mechanisms that regulate these processes. Age-related changes in the
growth hormone/IGF-1 axis are reviewed, and the effects of
moderate caloric restriction on the regulation of this axis are
presented. Finally, the potential effects of long-term moderate
caloric restriction on the regulation of cerebrovascular density
and cerebral blood flow are detailed, and its potential mediation
by growth hormone and IGF-1 are discussed. From these data
and recent publications demonstrating that IGF-1 is a potent
mitogenic agent and a risk factor for several types of cancer, we
conclude that growth hormone and IGF-1 have pleiotropic effects on aging and propose that neuroendocrine compensatory
mechanisms induced by moderate caloric restriction mediate
some of its beneficial effects.
The Growth Honnone/IGF-I Axis andAge
General.-Pure bovine growth hormone was first isolated
from the pituitary gland by Li and colleagues (34), and it was
subsequently shown to stimulate fatty acid mobilization, amino
acid uptake, DNA, RNA, and protein synthesis, and have a role
in cell division and tissue hypertrophy. Subsequent studies revealed that growth hormone secretion from the pituitary occurred in discrete pulses that increase after the onset of sleep
[(35) and see review by Corpas et al. (36)]. Although the precise function of this ultradian pattern remains unknown, the
pulsatile nature of growth hormone release/has been confirmed
. in every species examined to date and appears to be essential to
optimize biological potency of the hormone. Growth hormone
binds with high affinity to the growth hormone receptor, which
is found in tissues throughout the body; activation of its receptor stimulates the synthesis and secretion of hepatic IGF-1, a
small peptide (about 7.5 kD) structurally related to pro-insulin.
IGF-l circulates in the blood at high concentrations and stimulates DNA, RNA, and protein synthesis and is a potent mitogen
for many tissues (37). Because of the wide tissue distribution of
the growth hormone receptor, growth hormone may also have a
role in regulating the synthesis and secretion of IGF-1 from
many tissues, thereby directly influencing the paracrine activities of the hormone (38-40). Although it was initially proposed
that all of the actions of growth hormone were mediated
through IGF-l, several studies have provided relatively convincing data that growth hormone has direct effects on specific
tissues, and/or interacts with IGF-1 produced in tissue [(41,42);
refer to Figure 1).
In humans, growth hormone is released in pulsatile bursts
from the pituitary gland with the majority of secretion occurring at night in association with slow-wave sleep (35,43,44).
Similar pulses are observed in rodents, except that high-amplitude secretory pulses occur every 3.5 h in males (45) and hourly
in females. It was later discovered that the regulation of these
pulses involved at least two hormones released by the hypothalamus: growth hormone-releasing hormone (GHRH), which increases growth hormone release (46,47) and somatostatin,
which inhibits its release (48). The dynamic interactions between these hormones are responsible for high amplitude, pulsatile growth hormone secretion. It is generally believed that
somatostatin tone is dominant during trough periods and that
growth hormone is released in response to secretion of GHRH
and suppression of somatostatin (49). Both growth hormone
and IGF-1 inhibit growth hormone release in a typical feedback
relationship either directly at the level of the pituitary or by
stimulating somatostatin and/or inhibiting GHRH release (50).
Age-related changes in growth hormone and IGF-I.-lnitial
studies in elderly humans designed to investigate the relationship between growth hormone and physiological changes as-
PLEIOTROPIC EFFECTS OF GH AND IGF-I
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Figure I. The growthhonnone/lGF-1 axis. Growth hormone is secretedfrom
the anterior pituitary gland under the regulation of hypothalamic GHRH and
somatostatin. Growth honnonc circulates in blood, stimulates the liver to produce IGF-I , and influences the local production of IGF-I in target tissues. IGFI is respon sible for the anabolic effects of growth hormone on muscle and
many other target tissues. IGF-I activity is also regulated by several binding
proteins and tissue proteaseactivity(not shown).
sociated with age reported a decline in the ability of individuals to secrete growth horm one in respo nse to several stim uli,
including insulin-induced hypoglycemia and arginine administration (51). Subsequent studies revealed a loss of the nocturnal surges of growth hormone (35,52) and a decrease in plasma
IGF- l that paralleled the decline in growth hormone pu lses
(53,54). These early studies in humans have been confirmed
by numerous investigators [see review by Corpas et al. (36») .
It is now evident that the decline in high-amplitude growth
hormone secretion and plasma IGF-l concentrations is one of
the most robust and best characterized endocrine events that
occur with age (Figure 2).
Shortly after the documentation of decreases in growth hormone secretion in humans, studies confirmed that the amplitude of growth hormone pulses decreased with age in rodents
(55) and that these changes were associated with a decline in
plasma IGF-I (56). These studies immediately progressed to an
investigation of the mechanisms responsible for the decline in
growth hormone secretion. Several studies in both humans and
animal s documented a decline in in vivo pituitary response to
GHRH with age (57-59). However, over the next several years,
numerous studies attempting to detail the deficits within the
pituitary gland produced controversial results that ultimately
were attributed to either differential responses of older animals
to the pharmacological agents used to suppress endogenous
grow th hormone pulses during in vivo testing (60,61), or to
technical limitations in culturing anterior pituitary cells from
older animals (62).
Neuroendocrine reguLation.-Research efforts were eventually directed to an analysis of hypothalamic releasi ng and inhibiting hormones after studies revealed that (a) acute administration of somatostatin antiserum in vivo increased growth
hormone release identically in both young and old animals (63);
Figure 2. Age-related changes in plasma IGF-I in C57/BL6 male mice.
Plasma IGF-I is elevatedearly in life (maximum levels at 12-15 years of age in
humans and 25-60 days in rodents) and decreases substantially with increasing
age. As a result, the percent decline in IGF-I depends on the specific ages compared [adapted from Sonntag et al., 1992(22)1.
(b) passive immunization with somatostatin antiserum restored
the in vivo deficiency in pituitary response to GHRH (64); and
(C) stimulation of hypothalamic slices of old animals in a superfusion system released greater amounts of somatostatin than in
those of young animal s (65). More recently, we have found increased somatostatin peptide concentrations in pituitary extracts
from older animal s, again suggesting increased release of this
peptide from hypothalamic neurons (65).These results provided
compelling evidence that increased somatostatin tone may be
an important factor in the decline in growth hormone pulses
with age. These conclu sions were supported by research in humans, where administration of cholinergic agonists or arginine,
considered to preferentially inhibit somatostatin relea se (66),
was capable of increasing growth hormone secretion in older
individuals (67).
Although the previously mentioned studies did not address
the synthesis and release of GHRH, previous studies clearly indicated that a decline in this hormone is another contributing
factor in the decrease in growth hormone secretion with age. In
a series of experiments, investigators demon stated that GHRH
mRNA decreased with age (68), and that the feedback relationship between growth hormone and hypothalamic neurons is
impaired (69). In the latter study, growth hormone increased somatostatin mRNA and decreased GHRH mRNA, but GHRH
neurons were nonresponsive in older animals. Thu s, the data
suggest that dec reases in growth hormone secre tion with age
result from deficiencies within the hypothalamus involving regulation of both GHRH and somatostatin.
TIssue responsiveness to growth honnone.-Although a decline in the amplitude of growth hormone pulses is an important determinant of the decline in plasma IGF-l, more recent
studies demonstrate that growth hormone-induced IGF-l secretion is diminished in elderly individuals and suggest that resistance to the action of growth hormone may be a secondary contributing factor in the low plasma IGF- l concentrations (70). In
B524
SONNTAG ET AL.
rodents, a twofold increase in growth hormone receptors has
been observed with age, but this fails to compensate for the reduction in growth hormone secretion (71,72). A more detailed
investigation revealed that the K D and apparent size of the
growth hormone receptor were not altered, whereas the capacity of growth hormone to induce IGF-l gene expression and secretion was 40%-50% less in old than in young animals. These
results demonstrated that an irnpairrnent in growth hormone receptor signal transduction contributes to the decline in IGF-l in
both animals and humans.
The growth hormone receptor belongs to the cytokine family
of receptors, and its activation facilitates the association of the
receptor with JAK2 into a complex with subsequent phosphorylation of both proteins (73-76); see review by Roupas and
Herington (77). Several other intracellular proteins are subsequently phosphorylated, including mitogen-activated protein
kinase (MAP kinase), S6 kinase, and the STAT proteins (signal
transducer and activator of transcription). The result of growth
hormone receptor activation is an increase in c-fos, c-jun, serine
phosphatase inhibitor-I, and IGF-l gene expression (78).
Although diminished signal transduction has been described for
the insulin and IGF-l receptors in aged animals (79,80), no
study had addressed alterations in growth hormone receptor
signal transduction with age. Studies by Xu and colleagues (72)
reported that phosphorylation of JAK2 and the growth hormone
receptor complex were suppressed in aged rodents in response
to growth hormone, and that these decreases were accompanied
by a decline in MAP kinase activity. More recent studies
demonstrate that growth hormone-induced STAT3 activation
and nuclear translocation are also decreased with age (81).
Although these studies did not address the etiology of the decrease in JAK2 activity, they clearly indicated that diminished
growth hormone receptor signal transduction is a contributing
factor in the functional alterations in tissues with age.
Because the specific deficits in growth hormone signal transduction with age appear to be an early event in receptor signaling, we postulated that production of splice variants that are resistant to phosphorylation or a decrease in turnover of the growth
hormone receptor, perhaps resulting from oxidative damage to
the receptor molecule, contribute to the signaling deficit in old
animals. However, recent data from our laboratory demonstrate
that growth hormone receptor turnover in vivo increases with
age, and there are no obvious changes in receptor mRNA structure analyzed by RT-PCR (Xu and Sonntag, unpublished data).
Other mechanisms that potentially contribute to the decline in
growth hormone receptor signaling with age include point mutations, post-translationalmodifications in receptor protein, and enhanced phosphotyrosine phosphatase activity.Although the specific mechanisms for the decline in growth hormone signal
transduction are poorly understood, the deficiency in growth hormone signal transduction together with concomitant decreases in
growth hormone secretion appear to be the major factors involved
in the decline in plasma IGF-l with age.
Moderate Caloric Restriction and the Growth
Hormone//GF-l Axis
IGF-l.-Since protein synthesis is one of the intracellular
processes regulated by IGF-l, the hypothesis that increases in
protein synthesis observed in animals maintained under condi-
tions of moderate caloric restriction were mediated by attenuating the age-related decline in plasma IGF-I was assessed. Our
initial studies demonstrated that caloric restriction decreased
IGF-l approximately 40% in young animals and, with continued restriction, the age-related decline in IGF-l was attenuated
(82). However, contrary to our initial hypothesis, no differences
in plasma IGF-l levels were observed between old ad libitumfed and restricted animals (Figure 3). These results clearly indicate that the increases in protein synthesis observed in caloricrestricted animals were not dependent on the concentrations of
plasma IGF-l alone, and that other mechanisms for the increase
in protein synthesis in caloric-restricted animals (including type
1 IGF receptor density and insulin-like growth factor binding
proteins [IGFBP] levels) need to be pursued.
Subsequent analysis of type 1 IGF receptor density in tissue
revealed no consistent changes in ad libitum-fed animals with
age, but in response to caloric restriction there was a 1.5-2.5
fold increase in receptors in liver, heart, and skeletal muscle
from caloric-restricted compared to ad libitum-fed animals.
Because increases in receptor expression are generally associated with increases in tissue response, it was suggested that part
of the mechanism for increased protein synthesis in response to
moderate caloric restriction was related to enhanced tissue response to IGF-l (83) or alterations in the regulation of paracrine
(tissue) IGF activity.
IGF-l paracrine activity at the tissue level is regulated by (a)
the local production of IGF-l (84-89), (b) concentrations of
IGF receptors, and (c) the activity of binding proteins and specific proteases that degrade binding proteins releasing IGF-l to
interact with its receptor. In preliminary studies, we have found
that caloric restriction prevented the age-related decline in tissue levels of IGF-l analyzed after saline perfusion to remove
plasma IGF-l (Sonntag and colleagues, unpublished data).
Unfortunately, few data are currently available on the levels of
IGFBPs or protease activity in caloric-restricted animals.
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Figure 3. Age-related changes in plasma IGF-I in ad libitum-fed and caloric
restricted Brown Norway rats. Data indicate a reduction in IGF-l with age in
ad libitum-fed animals. In response to moderate caloric restriction, plasma IGFI decreases early in life and remains low throughout life span [from Breese et
al., 1991 (82)]. *p < .05 for ad libitum vs moderate caloric restriction.
PLEIOTROPIC EFFECTS OF GH AND IGF-l
Nevertheless, the results of studies to date are consistent with
the hypothesis that there is a differential regulation of tissue and
plasma IGF-l in response to caloric restriction (e.g., local expression of IGF-I is maintained whereas plasma IGF-I is substantially reduced). The specific consequences of the shift in the
source of IGF-I from the plasma to the tissue compartment are
not immediately apparent. Certainly, it has been demonstrated
that tissue maintenance and repair are facilitated by the
paracrine expression of IGF-l and, therefore, the paracrine expression of IGF-I into old age may be an important factor contributing to the maintenance of normal tissue function in response to specific stimuli. Concurrently, the reduction in plasma
IGF-l may be sufficient to diminish a generalized mitogenic
stimulus and thus influence the initiation and progression of
age-related pathologies (refer to Pleiotropic Effects section).
Growth hormone.-As noted previously, the age-related reduction in plasma IGF-I and cellular protein synthesis in ad
libitum-fed animals results in part from a decrease in the levels
of growth hormone secreted from the anterior pituitary gland
(55,57). Because of the decline in IGF-I in response to moderate caloric restriction observed in previous studies and the close
association of IGF-l with growth hormone concentrations (90),
the secretory dynamics of growth hormone in animals maintained under conditions of caloric restriction were not assessed
in our initial studies. However, our results demonstrating an increase in protein synthesis in caloric-restricted animals coupled
with increases in the number of tissue type 1 IGF receptors suggested that alterations in growth hormone secretion and the actions of growth hormone directly on paracrine IGF-I potentially could be an important mediating factor in the effects of
moderate caloric restriction. Consistent with previously reported studies, it was found that the amplitude of growth hormone secretory pulses decreased with age in ad libitum-fed animals and, as expected, the short-term consequence of caloric
restriction in young animals was a decline in the amplitude of
growth hormone pulses (Figure 4).
B525
Nevertheless, in older caloric-restricted animals, growth hormone pulses were similar to those in young, ad libitum-fed animals. The rise in plasma levels of growth hormone in old
caloric-restricted animals was unexpected, but both the number
of growth hormone pulses detected and the mean growth hormone concentrations increased substantially. From these studies, we concluded that after a period of adaptation to caloric restriction, animals exhibit increases in high-amplitude growth
hormone secretion that are maintained into old age. Because
plasma concentrations of IGF-I were diminished in the presence of increased tissue protein synthesis in these animals, the
studies again suggested the possibility that increased amounts
of circulating growth hormone may act directly at the tissue
level to maintain paracrine IGF-l activity. In either case, the
studies demonstrated that increased growth hormone secretory
dynamics are associated with longevity and may be part of the
mechanisms that enable these animals to maintain tissue function into old age.
Neuroendocrine regulation.-The aforementioned effects of
caloric restriction on growth hormone secretory dynamics led
us to use these animals as a model to assess potential neuroendocrine mechanisms for the decline in growth hormone with
age. In ad libitum-fed old animals, there was substantial evidence for increased somatostatin secretion, but further analysis
indicated a reduction in somatostatin mRNA in the hypothalamus of several strains of rats (91,92). These results raised an
important question regarding the mechanisms for the increase
in somatostatin secretion. To be translated into protein, mRNA
must attach to polysomes, and several lines of evidence suggested that this process can be regulated by specific intracellular proteins. In subsequent studies, we observed an increase in
somatostatin mRNA precipitating with the polysomal fraction
and an increase in the ratio of polysomalltotal somatostatin
mRNA. The changes in somatostatin mRNA initially occur
during mid-age, when growth hormone pulse amplitude decreases. Compared to the ad libitum-fed condition, the moder-
Old restricted
Old ad libitum
Young ad libitum
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Figure 4. Examples of growth hormone secretory dynamics in young ad libitum-fed (left), old ad libitum-fed (center), and old moderate caloric restricted (right)
Brown Norway rats. Blood samples were taken at 20-minute intervals from 0920 to 1640 h. Data indicate a significant reduction in growth hormone pulse amplitude
with age that is ameliorated by moderate caloric restriction [from Sonntag et al., 1995 (92)].
B526
SONNTAG ET AL.
thesis and release of the peptide; therefore, other mechanisms
must be considered. These data led us to conclude that intracellular factors regulate the association of somatostatin mRNA
with polysomes, and that altered regulation of these processes
with age results in increased synthesis of somatostatin.
Although our studies were not designed to address the specific
molecular mechanisms for the increased translational efficiency
with age, several possibilities exist; (a) there may be increased
synthesis and degradation of somatostatin mRNA with nascent
mRNA favored for translation; (b) aging animals may exhibit
diminished levels or activity of proteins or factors that normally
inhibit mRNA association with polysomes; and/or (c) there
may be increases in cytosolic facilitory factors that regulate association of somatostatin mRNA with polysomes. In recent
studies, several investigators have identified both cis and trans
acting regulatory elements in the 5'untranslated region of ferritin as well as other mRNAs that are important for the translational regulation of gene expression (96-105). These factors appear to facilitate or inhibit ferritin mRNA association with
polyribosomes, thus serving as an important regulatory site for
the synthesis of protein. Although there is no information
currently available on regulation of translational efficiency in
neuroendocrine systems, preliminary analysis of total and
polysome-associated somatostatin mRNA in response to hypophysectomy or eNS active drugs (Sonntag and colleagues,
unpublished observations) suggests that translational regulation
of somatostatin mRNA may be an important component of somatostatin synthesis and subsequently growth hormone secretion in adult animals.
ate caloric-restricted animals did not demonstrate a decline in
total somatostatin mRNA levels; when the fraction of somatostatin bound to the polysome was calculated, no age-related increase was apparent. The results of our studies again support
the conclusion that factors that regulate somatostatin gene expression have an important role in the regulation of growth
hormone secretion in aging animals. Furthermore, increased
association of somatostatin mRNA with the polyribosomal
fraction was closely related to a decline in growth hormone secretion in ad libitum-fed animals; caloric restriction, by delaying age-related changes in the translational regulation of somatostatin mRNA, appeared to maintain normal growth
hormone secretory patterns (Figure 5).
The regulation of growth hormone secretion is an excellent
example of a complex neuroendocrine feedback mechanism
compromised during normal aging. In young animals, in response to decreases in plasma growth hormone and IGF-I, total
somatostatin mRNA and peptide secretion decrease and GHRH
increases, resulting in the restoration of normal growth hormone levels (50,93-95). The decrease in somatostatin mRNA
in old ad libitum-fed animals in the presence of diminished
growth hormone and IGF-l concentrations is consistent with
the hypothesis that hypothalamic neurons from old animals are
capable of detecting the low levels of growth hormone and attempt to compensate by inhibiting somatostatin transcription
(resulting in the decrease in total somatostatin mRNA).
However, our previous studies on the regulation of growth hormone release in aged animals suggested that diminished transcription of somatostatin alone is insufficient to inhibit the syn-
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AGE (months)
Figure 5. Polyribsome-associated (left), total (center), and poly somal/total (right) somatostatin RNA in ad libitum-fed (6.) and moderate caloric restricted (e) aging
rats. The hypothalamus was dissected from each animal, homogenized in buffer, and aliquots separated for analysis of total and polysome-associated somatostatin
mRNA. Results indicated a decline in total somatostatin mRNA and an increase in polysomal mRNA in 25-month as compared to 6-month-old ad libitum-fed animals (p < .05). Data for polysomalltotal were calculated after correction to mRNA concentrations/tissue and are expressed as a percentage. A substantial increase in
polysomalltotal somatostatin mRNA was observed in 25-month as compared to 6- or 16-month-old ad libitum-fed animals (p < .05). The differences in somatostatin
mRNA were not observed in moderate caloric restricted animals. Data represent means ± SEM for 10 animals/group [from Sonntag et al., 1995 (92)].
PLEIOTROPIC EFFECTS OF GH AND IGF-I
Growth hormone signal transduction.-Because of our interest in the signal transduction properties of growth hormone
in older animals, we also assessed the effects of caloric restriction on JAK2 and growth hormone receptor phosphorylation
induced by growth hormone (Figure 6). Our results indicated
that moderate caloric restriction restored the deficient JAK2
and growth hormone receptor phosphorylation, MAP kinase
activity, IGF-1 mRNA levels, and IGF-I secretion in vitro in response to growth hormone (106). These studies did not address
the specific mechanisms responsible for the beneficial effects of
caloric restriction, but rather provided the first demonstration
that growth hormone signal transduction could be improved by
this regimen. These results raise the possibility that moderate
caloric restriction acts not only by increasing growth hormone
pulse amplitude, but also improves growth hormone signal
transduction resulting in enhanced growth hormone regulation
of!GF-1 paracrine activity.
Short-term caloric restriction ofold animals.-Although
previous studies demonstrated that caloric restriction beginning
at 4 months of age could prevent the age-related decline in
growth hormone pulse amplitude, the effects of short-term
moderate caloric restriction of aging animals remained unknown. Our rationale for these studies was to assess whether
the age-related decrease in growth hormone secretion was the
result of perrnanent alterations in the function of hypothalamic
neurons. We therefore analyzed growth hormone secretory dynamics in older animals that were initiated to the caloric restriction regimen at 26 months of age and had demonstrated a reduction in both growth hormone and IGF-l levels and a
decrease in insulin sensitivity. In response to 3 months of moderate caloric restriction, body weight in these animals decreased
by 30%. Concurrently, growth hormone secretory dynamics in-
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Figure 6. Effect of aging on growth hormone-induced growth hormone receptor phosphorylation in female C57BL6 mice. The liver was removed from
each animal and stimulated in media containing 2 nM rat growth hormone.
Tissue was homogenized in buffer and growth hormone receptor immunoprecipitated followed by Western blot analysis . Reactive bands were visualized
using ECL kits and quantified by densitometry. The average density of the
bands from different ages was corrected for receptor density in the respective
groups . Each point represents the mean ± SEM of six determinations. *p < .05
compared to old ad libitum-fed animals [from Xu and Sonntag. 1996 (208)].
B527
creased to levels indistinguishable fro m young animals,
whereas no increase in plasma levels of IGF-1 was observed
(Figure 7). Similar improvements were found for insulin sensitivity (Table 1). Older ad libitum-fed animals exhibited increased insulin levels compared to young animals during a glucose tolerance test, whereas old animals maintained for 3
months under conditions of moderate caloric restriction demonstrated marked improvements in insulin levels and demonstrated increased insulin sensitivity.These studies demonstrated
that (a) hypothalamic centers that regulate growth hormone secretion are not permanently impaired in old ad libitum-fed animals, and (b) age-related impairments in insulin sensitivity can
be reversed by caloric restriction. Although several possibilities
exist (including decreases in oxidative stress), one potential interpretation of these findings is that the reduction in adipose tissue mass decreases the secretion offactors that actively suppress growth hormone and insulin sensitivity. Cytokines such
as tumor necrosis factor-a (TNF-a), for example, have been
shown to suppress both growth hormone signal transduction
(107) and insulin sensitivity (108-112) in young animals. With
increasing age , we have evidence that TNFa secretion is increased (Xu and colleagues, unpublished data) and, if such factors can be shown to directly regulate the secretion of growth
hormone, important insight into one of the actions of moderate
caloric restriction would be attained (e.g., the reduction in adipose tissue and the corresponding reduction in secretory products from this tissue remove the inhibition of growth hormone
levels and hormone signal transduction).
Growth Hormone and IGF-I as Mediators ofthe Age-Related
Decline in Tissue Function
General.-Limited studies in rodents over a decade ago rcvealed that administration of growth hormone increases IGF-I
and restores tissue protein synthesis in old animals to levels
normally found in young animal s (22,57), sugge sting that the
age-related decline in skeletal muscle mass and function was
not solely related to intrinsic deficits within the tissue. Other reports were published demonstrating that growth hormone or
IGF-1 administration could partially reverse the decline in immune function (113,114), increase the expression of aortic
elastin (115) , and increase life span in rodents (114). The results from these studies were the first to indicate that the decrease in the concentration of growth hormone has clinical significance and may be responsible for the generalized catabolic
state that accompanies normal aging.
A number of investigators recognized the potential significance of decreases in growth hormone and have studied the effects of replacement therapy to older adults (116-1 18).
Although the specific populations under study were quite varied, it has generally been reported that administration of growth
hormone increases IGF-l , lean body mass, muscle mass, and
skin thickness and reduce s total body fat content in elderly humans. In addition, there are some reports of elevations in serum
osteocalcin (an osteoblast-produced marker of bonc formation),
urinary hydroxyproline (a marker for bone resorption), and nitrogen retention (a marker of protein synthesis), raising the possibility that growth hormone treatment may delay osteoporosis.
Interestingly, aerobic exercise training by elderly adults for I
year increas es the amount of growth hormone secreted over a
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Figure 7. Example of growth hormone secretory dynamics in young ad libitum-fed (left), old ad libitum-fed (center), and old short-term moderate caloric restricted
(right) Brown Norway rats (n =6/group) initiated to caloric restriction at 26 months of age for 3 months. Blood samples were taken at 20-minute intervals from 0920
to 1640 h and growth hormone was analyzed as described by Sonntag et al. (92). Data indicate a significant reduction in growth hormone pulse amplitude with age
that is ameliorated by short-term moderate caloric restriction [from Sonntag et al., 1996 (214)].
Table 1. Effectsof Short-Term CaloricRestriction on GlucoseTolerance
Young ad libitum
Old ad libitum
Old restricted
Glucose Sum
Insulin Sum
Insulin Response
Insulin Peak
1340.00 ± 144.82
1194.25 ± 92.58
1269.57 ± 89.10
275.03 ± 27.68
372.00 ± 26.15
232.60 ± 20.07*
119.26 ± 9.26
145.00 ± 9.65
114.51 ± 19.87
70.13 ± 6.76
84.05 ± 6.09
67.63 ± 5.83*
Insulin Fastiing
25.96±2.81
38.23 ±4.24
19.69 ± 1.99*
Notes: Blood samples were taken from 6 mo and 28 mo (ad libitum and moderate caloric restricted) Fischer 344XBN (PI) males at -2 and 5, 10, 15,20,30, and
40 minutes after infusion of 0.5 g/kg dextrose. Measurements for glucose sum are in milligrams per deciliter (mg/dl). Measurements for insulin are in microunits per
milliliter (J.1U/mL).
*p < .05 compared to old ad libitum-fed animals.
24-hour period (119), suggesting that the beneficial effects of
exercise training may be mediated, at least in part, by increasing growth hormone secretion. Our recent interests in the effects of growth hormone and IGF-l replacement have been focused in two areas: (a) the effects on growth hormone and
IGF-l on the cerebrovasculature, and (b) the effects of growth
hormone and IGF-l on brain function. These results (summarized below) suggest that critical interactions between these two
systems occur with age and may contribute to the decline in
cognitive function and increased risk of disease with age.
Cerebrovasculature and aging.-Previous studies indicate
that cerebral blood flow and capillary density decrease with age
in rodents, nonhuman primates, and humans and have the potential to be important contributing factors in brain aging
(120-126). Although the etiology of the decrease in cerebral
blood flow has not been determined, the decline did not appear
to be related to alterations in mean arterial pressure, as pressure
remains constant or increases with age. However, increases in
vascular resistance due to an age-related increase in arteriolar
vessel segment length between branches has been reported
(127). Hutchins and colleagues (128,129) demonstrated a decline in the total number of arterioles between the ages of 3 and
30 weeks in skeletal muscle, suggesting that a reduction in arteriolar density contributes to the decrease in blood flow. Because
an age-related decline in arteriolar density (or an increase in
vessel segment length) has the potential to decrease perfusion
pressure and tissue blood flow, we hypothesized that the decline in cerebral blood flow observed in aged animals may be
related to a decline in arteriolar density. This hypothesis has
been supported by our recent data demonstrating that an agerelated decrease of arteries, arteriolar-to-arteriolar anastomoses,
and venules occurs on the surface of the brain in rodents
(Figures 8, 9). In each case, we observed a decrease between
20% and 50% when young (4-6 months old) and old (25-32
months old) animals were compared. Because vasculature on
the surface of the brain has been demonstrated to be generally
representative of vasculature in other brain regions (130), we
concluded that the decline in blood flow with age resulted from
a decrease in the number of microvessels supplying blood to
the brain.
The maintenance of arteriolar density is a complex process
involving a number of growth factors. We and others have proposed that both growth hormone and IGF-I have an important
regulatory role in blood vessel growth and repair (89,131-133).
For example, blood vessels have receptors for growth hormone
and IGF-l, and several studies indicate that immunoreactive
IGF-l within vessels increases during periods of vessel growth
and repair (134,135). Furthermore, IGF-l has been shown to
potentiate the actions of several vascular growth factors (136).
Although it is well known that both plasma growth hormone
and IGF-I decrease and contribute to the decline in protein synthesis and vascular compliance that occurs in aged animals
(56,82,115,116), the role of these hormones in the age-related
PLEIOTROPIC EFFECTS OF GH AND IGF-I
A
B
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Figure 8. Left: Representative photographsof the cortical surface microvasculature in (A) young ad libitum-fed,(C) old ad libitum-fed, and (E) old caloricrestricted Brown Norwayrats, as seen through the cranial window. The entire parietal cranium has been removed. The cortex visible in this window extends from
the frontal to the occipital cortex. Vascular measurements were made over the
sensorimotor cortex. Results indicate a reduction in microvasculaturewith age
that is amelioratedby moderatecaloric restriction. Right: Pseudocolor-enhanced
autoradiograms of tissueconcentrations of [ 14CI iodoantipyrine in (B) young and
(D) old ad libitum-fed and (F) moderate caloric restricted animals under basal
conditions. Local cerebral blood flow was calculated by densitometricmeasurement of tissue concentrations of ['''CI iodoantipyrine after correction to autoradiographic standards,the concentrationcurve of blood [14C] iodoantipyrine, and
the appropriate constants according to the operational equation of the method
(MC= motorcortex;IXi = dentategyrus) [from Lynchet al., 1999(209)).
decreasein vascular density had not been assessed. Becausethe
decrease in cerebralblood flowappearsto be an importantfactor in brain aging, the regulation of vascular density by these
anabolichormoneshas the potentialto have therapeutic importancefor both vascularrepair and brainfunction.
Both growthhormoneand IGF-.J. havebeen reported to stimulate endothelialcell proliferation,tube formation, and angiogenesis in a number of tissues. Growth hormone, for example,
has been shown to stimulate angiogenesis in chorioallantoic
membranes of the chickembryo (133), whereas IGF-l has been
reportedto stimulate the growthof endothelial cellsin the retina
(137) and the proliferationof omental microvessel endothelial
cells (136). Similarly, IGF-l increasesangiogenesis and migration of endothelial cells in both rat aortic rings and bovine
carotid artery cells. Tube formation of carotid artery cells also
has been reported in response to IGF-I (138,139). In addition
to the apparentabilityof IGF-I to stimulate vasculargrowthin-
B529
dependently, several investigators reportthat the actions of other
growthfactorsincluding tissueplasminogen activator(tPA) and
hepatocytegrowth factor are facilitatedby IGF-l (136). These
studies supportthe hypothesisthat growthhormone and IGF-I
regulate vascular growth in vitro, and our recent data support
the conclusion that the decline in the secretion of these hormonescontributes to age-associated vascular deficiencies.
In our recent studies, the effects of growth hormone on vascular changeswithinthe agingbrain were assessedby administration of growth hormone to old animals for 30 days. As expected, vehicle-treated young and old animals exhibited no
changesin vasculardensityduring this period. However, a substantial increase in vascular growth in older animals treated
with growth hormone was observed (Figure 10). Although the
vascular growth observed in these animals represented only
small arterioles, and a complete restorationof vasculardensity
was not evident, the results provide compelling evidence that
growth hormone (and/orIGF-l) participate in the regulation of
vascular growth and contribute to the age-related declinein vasculardensity.
Obviously, vasculature has a crucial role in nutrient and
metabolic exchangewith tissues. However, severallaboratories
have reported the presence of specific growth factors that are
producedin the peripheralvasculature, suggestingthat the vasculature exerts a trophic influence on surrounding tissues.
(89,140,141) . In our own studies, we haveobserved that the microvasculature throughout the brain is a major site of IGF-I
production(Figure 11). Withage, totalIGF-l concentrations in
brain decrease by 30%-40% and, because of the high concennations of IGF-I in the microvessels, we haveproposedthat the
reduction in vessel density is one of the factors that contribute
to the decline in brain IGF-l levels. The production of these
growthfactors by the vasculature may have importantimplications for the regulationof brain function during aging. Because
of the importantinterrelationships between brain function and
cerebrovasculature and the presenceof IGF-l as a potential mediator in both neuronaland vascularfunction,detailedanalysis
of the mechanisms responsible for the age-related decline in
vascular function meritsfurtherinvestigation.
Moderate caloric restriction and microvasculature.-Com-
parison of vascular density on the surfaceof the brain between
ad libitumand moderate caloric-restricted animalsrevealed that
the age-related decline in vascular density was almost completelyamelioratedby moderatecaloric restriction. The effects
of chroniccaloricrestriction were also evidenton localcerebral
blood flow. Using [14C]iodoantipyrine, analysis indicated an
age-relateddecrease in blood flow in most areas of cortex and
hippocampus (approximately 30%), and this decreasewas partially prevented by moderate caloric restriction. Preliminary
data also suggested that moderate caloric restrictionprevented
the age-related decline in brain IGF-Ilevels. Althoughthe specificconsequencesof maintenanceof vasculardensityinto old
age are unclear, we suspectthat the increasein vasculardensity
results in a rise in regional blood flow and vascular-derived
IGF-l that has the potential to have trophic effectson the brain.
The vasculareffectsof moderatecaloricrestriction were also
evidentwhen older animalswere fed 60% of the ad libitumdiet
for 30 days (Figure 12). During this period, there was a growth
of small arteriolesthat was similarto that observedin response
SONNTAG ET AL
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Figure 9. Summary of arteriolar (left) , arteriole-to-arteriole anastomotic (center). and venular endpoint (right) density in mal e Brown Norway rats . Data represent
mean :l: SEM for 18 young . 14 middle-aged , and 13 old animals. Results indicate that arteri olar and arteriolar-arteriolar anastom ot ic density dec reases with age ( p <
.05) Ifrom So nntag et al.• 1997 (210)].
*
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Figure 10. Effects of bovine growth hormone (25 ug/kg, twice dail y ) or vehicle administration for 35 day s on changes in arteriolar density in 30-month-old
Fischer 344 x Brown Norway rats. Data are expressed as the percent increase
in arteriolar den sity from baseline and repres ent mean ± SEM of 10 (gr owth
hormone-treated) and 7 (saline-treated) animals/group. No vascular growth was
observed in 8- or 30- m ont h-o ld sali ne -treate d an im als [fr om Sonnta g et al.,
1997 (210)1. *p < .01.
to growth hormone administra tion noted previously. These findings demonstrate the occ urre nce of significant age- related decreases in vasculature that can be ameliorated by either caloric
restriction or growth hormone. These results raise the possibility that moderate caloric restriction influences neuronal function in part via its effects on vascular density and the secretion
of vascular-derived IGF- 1. The similarities betwee n the actions
of growth horm one administration and caloric restriction also
suggest that some of the effects of ca loric restriction may be
mediated by growth hormone. Although this relationship is correlational at the present time, further studies to assess the relationship between these factors are clearly warranted.
CNSjunctio ll.-Although data on growth hormone's direct
action on neuronal function are extremely limited, many investigators have prov ided convinc ing evidence that IGP- l and
IGF-2 have an important role in this regard (86,88 ,142) . IGFs
have been reported to stabilize tubulin mRNA (143), stimulate
DNA and RNA synthesis (142, 144-146), stimulate neurite formatio n (14 7- 149) , enh ance oligo dendroc yte prol iferation
( 150- 153), increase survival of neu ron s and glia in culture
(154), increase neuromu scular synaptogenesis (155), and have
an import ant role in neuronal rep air (156) . More recent evidence suggests that IGF- l partici pates in the regulation of calcium and increases the expression of the proto-oncogene c-fos
(157,158). Although there is a large volume of data supporting
a role for IGF-l in brain function, there are few in vivo studies
of the actions of IGF-I , in part, because of the lack of appropriate models to manipulate IGF-l levels.
While growth hormone and plasma IGF- I appear to have a
role in brain function [see review by Hepler and Lund (159)],
the sources of IGF- I that influence the brain are less clear. The
maj ority of evidence suggests that grow th hormone does not
cross the blood-brain barrier although it is known that hypophysectomy (wh ich decreases both grow th hormo ne and plasma
IGF-I ) dec reases IGF -1 mR NA in brai n, and concentra tions
can be restored by growth hormone administration (160). In addition, recently published studies suggest that administration of
growth hormone raises brain conce ntrat io ns ofIGF-1 (16 1),
and peripheral injections ofI GF- I have been shown to protect
neurons from cell death after ischemic injury (162). Evidence
PLElaTROPIC EFFECTS OF GH AND IGF-}
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.-
Figure II . Localization of IGF-I mRNA in rat cerebral cortical microvasculature demonstrated by in situ hybridization. Left : bright field photomicrographs for three different arteriole s in the sensorimotor cortex that express
above -background levels oflGF-1 mRNA. Right: Pseudocolor-enhanced images of pixel density analysis of these same three vessels. Magnification (A) =
300x, (B) = ISOX, (C) = ISOX [from Sonntag et aI., 1999 (210).
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Figure 12. Effect of short-term moderate caloric restriction (60% of ad libitum) on percent increase in vascular density in young and old Brown
NorwayXF344 male rats . Animals were initiated to caloric restriction at 26
months of age for 3 months. Vascular density was analyzed as des cr ibed by
Sonntag et al., 1997 (210). Results indicate that caloric restriction increases vascular density on the cortical surface of the brain (From Poe B, Lynch C, Cooney
P, Brunso-Bechtold JK, Sonntag WE , Hutchins PM. Caloric restriction increases cortical microvasculature in aged animals. Soc Neurosci Abstracts.
1996;22:1236).
B531
supports the conclusion that plasma IGF-1 is actively transported
through the blood brain barrier (163), but the specific mechanism
for this process is poorly understood. Because IGF-l is also produced in endothelial and smooth muscle cells (87,89,140), the
possibility exists that both plasma IGF-1 and vascular-derived
IGF-l (possibly under the regulation of growth hormone and/or
IGF-l) are important sources of IGF-I in the brain.
As a large body of evidence supports the conclusion that
IGF-l is an important neurotrophic factor that decreases with
age, we undertook studies to assess the effects of intracerebroventricular IGF-l replacement on learning and memory as
well as several biochemical measures linked to cognitive ability. Our studies revealed that administration of icv IGF-l for
28 days to old animals increased both working and reference
memory using the Morris water maze [Figure 13; (164)].
In addition, preventing the age-related decline in growth hormone pulse amplitude and plasma IGF-1 by daily injections of
[D-Ala2]GHRH for 18 months prevented the age-related
decline in reference memory. In fact, performance of old [DAla2]GHRH animals on the reference memory task was indistinguishable from young animals. Although the specific mechanisms for the improved performance in response to these
anabolic hormones are still under investigation, we have found
that aged animals exhibit a decrease in specific subtypes of the
NMDA receptor (NMDA R2a-c), and that IGF-1 reverses
these age-related deficits (165). Similarly, IGF-l appears to reverse the age-related decrement in D'-induced receptor activity
that is evident in older animals (166). Because both NMDA
and dopamine receptors have been strongly implicated in the
process of memory acquisition (167-171) , it is tempting to
speculate that the behavioral improvement noted in response to
IGF-l is based, in part, through effects on these neurotransmitter systems.
The effects of moderate caloric restriction on brain levels of
IGF-l, cerebrovascular density, and blood flow led us to compare reference memory between caloric-restricted and ad libitum-fed aged animals. We observed that moderate caloric restriction prevented the age-related decline in reference memory,
which is in close agreement with several other studies indicating that this regimen prevents the age-related decline in sensorimotor function and cognitive ability in mice( 172, 173).
Although the studies conducted to date do not allow us to draw
definitive conclusions, the importance of cerebral blood flow
and the trophic actions of vascular-derived IGF -l lead us to
speculate that modest deficiencies in blood flow to the aging
brain resulting from a rarefaction of vasculature may contribute
to the reduction in neurological function and possibly agerelated pathologies (refer to Figure 14 for a general summary of
the effects of age on the growth honnone/lGF-1 axis).
Pleiotropic Effects ofGrowth Honnone/lGF-l
General.-As previously noted, the increase in growth hormone in moderate caloric-restricted animals provides access to
the energy source of free fatty acids stored in adipose tissue.
Growth hormone and other hormones including cortisol and
circulating catecholarnines stimulate hormone sensitive lipase
and increase the availability of nutrients for tissues-representing an important physiological adaption to moderate caloric restriction . Similarly, the decline in anabolic hormones such as
SONNTAG ETAL
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Figure 13. Effects of intracerebroventricular administration of IGF-I or vehicle on reference memory in Brown Norway rats. Data are expressed as percent of control for both the time spent in a 40 cm annulu s surrounding the platform (left) and the number of times the platform location was crossed (rightj duri ng the probe trial.
Data indicate a significant decrease in reference memory with age that is ameliorated by admini stration ofIGF- J [adapted from Markowska et al., 1998 (164) ]. O-Sal
= old saline-injected; O-IGF = old IGF-I -injected .
*p< .05.
Figure 14. Summary of alte rations in the growth horm one/lGF-1 axis in yo ung (left) and old (ce nter) ani mals and in response to moderate caloric restriction
(right). Growth hormone secreto ry pulses and tissue response to growth hormone decrease with age in ad libitum-fed animals, resulting in a decline in plasma IGF-I .
Moderate caloric restriction increas es growth hormone pulse am plitude and decreases plasma IGF-l . The increase in growth hormone increases paracrine activity of
IGF-I in tissue.
IGF-I is adapti ve in that cellular growth is minimized while
limited nutrients are targeted for essential cellular functions, including tissue maintenance and repair. Thu s, the condition of
moderate caloric restriction produces unique alterations in the
endocrine system- not only in the regulation of growth hormone and IGF- I, but also the regulation of cortisol, insulin,
catecholamines, and numerous other factors that regulate homeostasis (174).
Growth hormone/IGF-I and the physiological manifestations ofaging.-Research over the past 15 years has clearl y
demon strat ed that administration of growth hormone and/or
IGF- I rever ses many of the physiological changes in tissue
function, and we have propo sed that the aging phenotype result s, in part, from chronic, multiple horm one deficiencies.
Although the concept of aging as hormone deficiency has not
been readily accepted, the capacity of estrogen replacement to
ameliorate the age-related increa se in cardiovascular disease,
the decrease in bone mass, and mo st recently the decreases in
cognitive function in women clearly suggests that hormone deficiency is an important contributing factor in many age-related
processes. The ability of growth hormone to increase intracellular protein synthesis, cognitive function, skin thickne ss, bone
mass, immune function, and vascular density in older animals
and hum ans again provides comp elling evidence that the decline in the se hormones has important physiological con sequences for the developm ent of the aging phenotype. As intriguing as the possibilities are , however, the long-term
beneficial effects of replacement therapy have been questioned
because increases in anabolic hormon es have the potential to
incre ase the risk of path ology and therefore may decrease,
rather than increase, life span.
B533
PLEIOTROPICEFFECTS OF GH AND IGF-l
Growth honnone/IGF-l, tissue pathology and life span.An excellent example of anabolic hormones increasing risk of
pathology is recent studies that have provided a link between
the incidence of a number of cancers and the expression of
IGF-l. Using in vitro models, it has been recognized for some
time that IGF-l is a potent mitogenic factor that increases the
transition from G 1 to S phase in the cell cycle. In cell culture,
serum supplementation is generally necessary for cell survival.
Serum contains high quantities of IGF-l, and antibodies
against IGF-l are able to inhibit the ability of 5% serum to
stimulate DNA synthesis in Balb/c3T3 cells (175). In addition,
Balb/c-3T3 cells transfected with IGF-1 and the type 1 IGF receptor grow in serum-free media (176), and antagonists to the
IGF receptor inhibit cellular growth (177). It also appears that
other growth factors, including platelet derived growth factor
(PDGF) and epidermal growth factor (EGF), increase cellular
IGF-1 synthesis, and it has been proposed that their effects
may be mediated in whole, or in part, through the IGF-1
system.
Similar stimulatory effects of IGF-1 on cellular growth have
been reported in vivo. Numerous human cancers and transformed cell lines produce IGF-1 or its receptor [see review by
Werner and LeRoith (178)], and overexpression of the type 1
IGF receptor in 3T3 fibroblasts leads to the formation of tumors
in nude mice (179). As expected, passive immunization with
antibodies against the type 1 IGF receptor inhibits the proliferation of numerous cell types and cancers (180-188). As expected, transgenic rodents expressing high levels of human
growth hormone and subsequently plasma IGF-1 exhibited an
increase in mammary tumors; however, degenerative changes
that appear to resemble aging were also apparent (189). In humans, elevated IGF-1 levels have been demonstrated to be a
risk factor in breast cancer (190-193), lung cancer (194-197),
and prostate cancer (198). These studies demonstrate an important link between plasma IGF-1 and the appearance of tissue
pathologies and raise the possibility that a reduction in IGF-1 or
IGF-l activity may induce a selective advantage by delaying or
preventing age-related pathologies.
It is interesting to note that the substantial decrease in IGF-1
observed in caloric-restricted animals early in the life span is
associated with a decline in the number of pathologies compared to ad libitum-fed animals. Comparison of pathological
changes in various cohorts appears to indicate that the beneficial effects of caloric restriction are detectable early in the life
span (14). Additionally, one of the strongest predictors of life
span is body weight at 10 months of age, which is known to be
highly dependent on circulating levels of IGF-1. Although
more research will be required to establish a link between the
decline in IGF-1 and decreased pathological risk in moderate
caloric-restricted animals, the implication of these results is
that early exposure to high concentrations of IGF-1 or a cumulative exposure to IGF-1 during the early phase of life initiates
pathological changes in tissues that are manifest at later ages
(Figure 15).
The potential of IGF-l as a risk factor for a number of agerelated pathologies raises the question as to whether IGF-1 has
a role in determining life span of the organism. However, studies designed to assess this relationship are difficult to interpret
in part because of the close relationship between growth hormone and IGF-l and inherent difficulties with all of the mod-
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Figure 15. Proposed theoretical relationship between IGF-I and the incidence of pathologies during the life span in ad libitum-fed and moderate caloric
restriction animals. The decrease in IGF-I induced by caloric restriction
decreases the risk of pathological events within tissues and increases life span.
els. For example, studies in mice suggest that immune function
and life span could be improved by administration of low
doses of growth hormone that result in mildly elevated IGF-l
levels (199). However, subsequent studies by Kalu and colleagues (200) using several strains of mice and rats suggested
that administration of physiological doses of human growth
hormone to animals beginning at 17 months of age (resulting
in a modest increase in plasma IGF-l) did not result in a general increase in life span. At the same time, they did not observe an increase in the number of pathologies or a shortening
of life span. Both of these studies used human growth hormone
in rodent models that is known to exhibit both sommatogenic
and prolactogenic properties (201-204). In our own laboratory,
we have observed that chronic injections of [D-Ala2]GHRH to
increase endogenous levels of growth hormone in rodents have
little effect on life span or age-related pathologies (205). In another study, which used transgenic animals producing aphysiological hormone levels, ithas been suggested that high concentrations of growth hormone and IGF-I shorten life span in a
dose-dependent manner (189). In addition, low levels of
growth hormone and IGF-1 (as seen in dwarf mice) are associated with increased life span. The latter results are somewhat
limited in that the dwarf mice used in the study were deficient
in several hormones including growth hormone, prolactin, and
thyroid-stimulating hormone (206,207). Nevertheless, these
studies are consistent with previous results indicating that high
levels of IGF-1 are a contributing factor in age-related pathologies, and they raise the important issue related to interactions
of body size with life span (possibly independent of the contribution of growth hormone and IGF-1). Obviously, further research is necessary to resolve the specific role of these hormones in the onset of age-related pathologies and life span.
Future studies will require the development of transgenic models that isolate the actions of each hormone and use concentrations that are physiologically relevant for aging studies.
B534
SONNTAG ETAL
Conclusion
There is a large body of evidence that alterations in the regulation of the growth hormone/lGF-1 axis have significant effects on age-related alterations in physiological function,
pathology, and eventually life span. With age, both growth hormone and IGF-1 decrease and administration of these hormones ameliorates the effects of aging on numerous tissues including the central nervous system (reversing the age-related
decline in cerebrovascular density and cognitive function). In
response to moderate caloric restriction, important endocrine
compensatory mechanisms are invoked that represent an important adaptive process to maintain blood levels of glucose.
These adaptations include an increase in growth hormone pulse
amplitude, a decline in plasma levels of IGF-1, and possibly a
shift to increase the paracrine activity of IGF-1. These alterations in the growth hormone/lGF-1 axis can be induced in
young or old animals and appear to have important physiological effects on tissue at any age.
Although the age-related decreases in growth hormone and
IGF-1 contribute to the aging phenotype, IGF-1 is also linked
to a number of pathologies and appears to be an important risk
factor for breast, lung, and prostate cancer. The relationship between IGF-1 and tissue pathology has led us to speculate that
the decrease in plasma IGF-1 in response to moderate caloric
restriction minimizes the mitogenic stimulus to tissues and contributes to the reduction in age-related pathologies and increased life span observed in this model. Because IGF-1 has
also been demonstrated to be an important trophic factor for tissue maintenance and repair, the rise in growth hormone, which
may drive paracrine IGF-1 activity, may be equally important.
On the contrary, studies indicating that cellular replicative potential is reduced in transgenic mice overexpressing growth
hormone and IGF-1 (212) and that moderate caloric restriction
increases replicative potential (213) provide additional associations between the effects of IGF-1 and biological aging. At the
present time, it is unknown whether high levels of IGF-1 during
specific periods in the life span or throughout life contribute to
the induction of age-related pathologies and the regulation of
cellular replicative potential.
The plethora of effects induced by moderate caloric restriction suggests that other endocrine and nonendocrine systems
may also contribute to the beneficial effects of this regimen on
age-related pathologies and life span. Alterations in plasma levels of cortisol, catecholamines, and insulin, as well as other
peptide hormones including leptin and neuropeptide Y, may be
critical in mediating the response of these animals to fasting
and thus potentially have a role in the effects of moderate
caloric restriction. Similarly, the decline in blood levels of glucose and the reduction in oxidative stress have been demonstrated in response to caloric restriction of a number of species
and may provide independent or overlapping mechanisms that
contribute to the health of these animals. Nevertheless, compensatory changes in the endocrine and neuroendocrine systems appear to have a key role in the processes of moderate
caloric restriction and underscore the significance of age-related
changes in the neuroendocrine system.
ACKNOWLEDGMENTS
This research was supported by grants AG-07752 and AG-11370 from the
National Institute on Aging.
Address correspondence to Dr. William E. Sonntag, Department ofPhysiology and Pharmacology, Wake Forest University School of Medicine, Medical
Center Boulevard, Winston-Salem, NC 27157-1083. E-mail: wsonntag@
wfubmc.edu
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Received September 1,1998
Accepted February 17,1999