Abstract. Background: Calorie restriction (CR) inhibits
carcinogenesis and delays aging. Some anti-carcinogenic effects
of CR are mediated by decreased circulating insulin-like growth
factor-1 (IGF-1); however, IGF-1 also plays an important role
in regulating growth and bone density. Materials and Methods:
We quantified tradeoffs involving the CR/IGF-1 axis in
C57BL/6 mice by examining body composition and bone
characteristics in ad libitum fed, 20, 30 or 40% CR mice that
received placebo or recombinant murine IGF-1 delivered with a
time-release pellet. After 26 days, carcasses were scanned with a
PIXImus II dual-energy X-ray absorptiometer. Results: CR
reduced body weight and percent body fat and had non-linear
effects on bone density. IGF-1 restored bone density to control
levels or greater in the CR mice. Conclusion: Cancer prevention
efforts based on CR and down-regulation of the IGF-1 pathway
will require consideration of deleterious effects on bone.
Calorie restriction (CR) is known to delay aging and
carcinogenesis in rodents as well as in several other animal
models (1-3). These results have led to intense interest in
identifying mechanisms underlying the beneficial effects of
CR and in developing preventive and treatment strategies
based on these mechanisms (4, 5). Several lines of evidence
show that modulation of the insulin-like growth factor-1
(IGF-1) pathway influences carcinogenesis and is a
mediator of at least some of the cancer preventive effects of
CR. CR animals have lower serum levels of IGF-1 (6) and
increased latency and decreased number and size of tumors
(7). Compellingly, restoration of IGF-1 levels in CR animals
by exogenous administration of IGF-1 accelerates their
otherwise delayed tumor development (7). In vitro studies
have demonstrated that IGF-1 stimulates the growth of
numerous cancer cell lines (8-10), and a number of
epidemiological studies indicate that serum IGF-1 levels are
positively associated with risk of colon, breast and prostate
Calorie restriction and changes in body weight are also
known to influence bone characteristics. In humans, bone
density is positively correlated with body weight (14, 15),
and weight loss results in decreased bone density (14). In
rodents, experimentally imposed CR reduces bone density
(16, 17). IGF-1 is believed to be part of the mechanistic
pathway linking body weight and bone density (18, 19). For
example, MIDI mice have circulating IGF-1 levels
diminished by 60% and have reduced bone density; IGF-1
treatment increases their bone density (20). Together, these
observations suggest that efforts to slow aging or prevent
cancer via CR or modulation of the IGF-1 axis must take
into account the effects of reduced levels of IGF-1 on bone.
In the present study, we examined the effects of several
levels of CR with and without treatment with recombinant
murine IGF-1 on body composition, bone characteristics
and serum IGF-1 levels, in order to further characterize
*The publisher acknowledges the right of the U.S. Government to
retain an exclusive, royalty-free license in and to any copyright
covering this article.
#These two authors contributed equally to this project.
Correspondence to: David Berrigan, Applied Research Program,
Division of Cancer Control and Population Sciences, National
Cancer Institute, Executive Plaza North MSC 7344, Room 4009A,
Bethesda MD 20892-7344, Tel: 301-451-4301, Fax: 301-435-3710,
Key Words: Bone mineral density, calorie restriction, insulin-like
growth factor-1, mice, cancer prevention.
in vivo 19: 667-674 (2005)
Phenotypic Effects of Calorie Restriction and
Insulin-like Growth Factor-1 Treatment on Body
Composition and Bone Mineral Density of
C57BL/6 Mice: Implications for Cancer Prevention*
DAVID BERRIGAN1#, JACKIE A. LAVIGNE1#, SUSAN N. PERKINS1,
TIM R. NAGY2, J. CARL BARRETT1,3and STEPHEN D. HURSTING1
1National Cancer Institute, Bethesda MD 20892;
2Department of Nutrition Sciences, University of Alabama at Birmingham, Birmingham, AL 35294;
3Novartis Institutes for Biomedical Research Inc., Cambridge, MA 02139, U.S.A.
potential tradeoffs between beneficial effects of CR and
reduced IGF-1 levels versus their potentially deleterious
effects on bone characteristics.
Materials and Methods
The mice were maintained according to the guidelines of the Animal
Care and Use Committee of the National Cancer Institute, and the
committee approved this study. Five-week-old female C57BL/6NCr
mice (Charles River, Frederick, MD, USA) were singly housed and
received water ad libitum (AL). In the first week after receipt, mice
were fed AIN-76A diet (Bio-Serv Corp., Frenchtown, NJ, USA) AL,
and food consumption was measured. All mice then received a time-
release pellet (Innovative Research, Sarasota, FL, USA) containing
placebo, recombinant murine IGF-1 or recombinant human IGF-1
obtained from Peprotech, Inc. (Rocky Hill, NJ, USA). Pellets were
implanted subcutaneously under isoflurane anesthesia along the
dorsal thoracic midline about 3 cm anterior of the hip via a small
incision made on the upper dorsal skin. The study diets were started
three days after pellet implantation.
For the main study, 45 mice were randomized (n=5 per group)
to one of nine groups: i) AIN-76A diet AL + placebo pellet, ii)
AIN-76A diet AL + murine IGF-1 (20 Ìg/day), iii) 20% CR +
placebo pellet, iv) 20% CR + murine IGF-1 (20 Ìg/day), v) 30%
CR + placebo pellet, vi) 30% CR + murine IGF-1 (20 Ìg/day), vii)
30% CR + human IGF-1 (20 Ìg/day), viii) 40% CR + placebo
pellet, ix) 40% CR + murine IGF-1 (80 Ìg/day). The human IGF-1
treatment group was included to determine if mouse and human
IGF-1 had different effects. The AL-fed groups received AIN-76A
diet AL. Mice in the 20% CR groups received a daily aliquot of
AIN-76A diet equal to 80% of the mean daily AL consumption
measured in the previous week. CR mice (30% and 40%) received
modified versions (Bio-Serv. Corp) of the AIN-76A diet
formulated such that, when provided as daily aliquots equal to 70%
or 60%, respectively, of the mean daily AL consumption, the
reduction in calorie intake was entirely due to carbohydrates;
intakes of all other nutrients were equivalent to those in the AL
group (Table I) (21). Body weight and food consumption data were
recorded weekly and at study termination after 26 days on the
diets. The first body weight measurement was obtained 2 days after
dietary treatment began and 5 days after pellet implantation. On
the final day of the experiment, mice were sacrificed under
continuous CO2/O2anesthesia and blood collected. Serum,
selected tissues and necropsied carcasses were frozen and stored at
–80ÆC for subsequent analyses.
We examined the time-specific effects of IGF-1 pellets in a
second cohort of 15 mice randomized (n=5 per group) to one of
three groups: i) AIN-76A diet AL + placebo pellet, ii) 30% CR +
placebo pellet, and iii) 30% CR + murine IGF-1 (20 Ìg/day). Blood
was collected via the retroorbital sinus or saphenous vein at baseline,
24 and 72 h after pellet insertion and at days 2, 4, 7, 14, 21 and 28 of
CR (72 h after pellet insertion corresponded to day 1 of CR); the
serum was frozen and stored at –80ÆC until assay of IGF-1.
Fat weight, lean weight, bone mineral density (BMD) and bone
mineral content (BMC) were determined using dual-energy X-ray
absorptiometry (DXA) (GE Lunar Piximus II, Madison, WI, USA).
Replicate measurements (2-3) were made on each individual
animal. In brief, necropsied carcasses were placed on the specimen
tray and repositioned after each scan (22). After scanning, GE-
supplied software (version 1.46) was used to extract data. Animals
were weighed prior to and after necropsy and prior to scanning. An
adjusted fat weight was calculated as the fat weight estimated for
the necropsied carcass plus fat weight contained in tissue removed
during necropsy. The fat content of necropsied tissue was estimated
by assuming that these tissues contained the same average
percentage fat as animals from the calorie-restricted treatment. This
procedure is a conservative approach that is likely to underestimate
true fat contents. Lean weight was calculated by subtracting
adjusted fat weight from weight prior to necropsy. Lastly, we
estimated bone density in the tibia alone and in the vertebrae alone
by selecting the left tibia as the region of interest (ROI) or the
vertebrae from the midpoint of the pelvis to the point where the
ribs met the vertebrae for each mouse. We validated our DXA
measurements of 40 necropsied carcasses (human IGF-1-treated
group not included) using gravimetric and chemical (Soxhlet)
extraction (22, 23).
Serum IGF-1 was measured with a rat radioimmunoassay (RIA)
IGF-1 kit that recognizes both rat and mouse IGF-1 (Diagnostic
Systems Laboratories, Inc., Webster, TX, USA). Serum IGF-1 in
mice from three groups (AIN-76A diet AL + placebo pellet, 30%
CR + placebo pellet, and 30% CR + human IGF-1) was also
measured with a human IGF-1 RIA that recognizes both rodent
and human IGF-1 (Nichols Institute Diagnostics, San Juan
Capistrano, CA, USA). Average values for two determinations
made on a single aliquot of serum from each animal are reported.
Total IGF-binding protein (IGFBP) activity was assessed using a
slot-blot assay (24). In brief, 2 Ìl of serum was applied to a
nitrocellulose membrane (0.45-Ìm pore size) (Schleicher and
Schuell, Keene, NH, USA), by slot-blot manifold (Schleicher and
Schuell); the membrane was probed with 1.5x106cpm of 125I-IGF-2
(Amersham Biosciences, Piscataway, NJ, USA) and exposed to
XAR film (Kodak, Rochester, NY, USA). Samples were assayed
on 3 independent blots and the banding signal intensity was
quantified with a densitometer. Total IGFBP activity was expressed
as percent of the average value for the AIN-76A diet AL +
placebo pellet group.
in vivo 19: 667-674 (2005)
Table I. Diet composition.
Ad Libitum (AL)
& 20% CR1
Salt Mix, AIN-76A
Vitamin Mix, AIN-76A
Choline Dihydrogen Citrate
1The 20% CR animals received daily aliquots of the same diet consumed
by AL-fed mice equal to 80% of their average daily food intake.
230% and 40% CR mice received the appropriate CR diet in daily aliquots
equal to 70 or 60% of the average daily food intake of AL-fed mice.
Analysis of variance and analysis of covariance were used to
assess CR and IGF-1 treatment effects. Means from univariate
analyses were compared using Tukey’s HSD test. Analysis of
covariance allows the inclusion of weight as a covariate in the
statistical analysis of the association between bone density, fat
content and treatment effects (25). All analyses were performed
using SAS JMP Version 5.0 (SAS Institute Inc., Cary, NC, USA).
These tests were followed by standardized linear contrasts
comparing CR to the AL-fed controls, IGF-1-treated groups to
their respective CR controls, and IGF-1-treated groups to the
AL-fed controls. These linear contrasts use a t-test to determine
whether control and CR groups differ, without regard to the level
of CR. Presence or absence of dose-response relationships between
CR and body composition were tested with linear regression
models. All tests were two-sided; probability values less than 0.05
were considered significant. Lastly, IGF-binding protein activity
was analyzed using ordinal logistic regression because the data are
reported as ratios.
Validation of DXA on necropsied animals. Precision was
determined by calculating the mean intra-individual
coefficient of variation (CV) for our repeated DXA
measurements. Mean % coefficients of variation for fat
weight, bone mineral content and bone mineral density were
6.2%, 4.6% and 1.6%, respectively. Accuracy was
characterized by performing analyses of covariance with
DXA-estimated parameters as dependent variables,
chemical carcass analysis estimates as independent
parameters and experimental treatments as covariates. We
report regression coefficients for the association between
DXA and chemical methods (CHEM) for estimates of body
fat (g) and total body ash. Here, we used the uncorrected
estimate of fat weight from the necropsied carcasses in
order to match the results from chemical extraction. The
following equations are presented as: DXA measurement=a
+ b* Chemical Measurement; 95% confidence intervals are
given in parentheses. For fat weight (g), DXA Fat
Weight=1.74 (1.2-2.2) + 0.93 (0.7-1.1) * CHEM Fat Weight
(n=40, r=0.98). For bone mineral content, DXA
BMC=0.17 (0.0-0.3) + 0.61 (0.2-1.1) * CHEM Bone Ash
(n=40, r=0.96). These r values are comparable to results
Berrigan et al: Calorie Restriction and IGF-1 Effects on Mouse Bone and Body Composition
Table II. Body composition and bone characteristics of ad libitum (AL)-fed, CR and IGF-1 treated C57BL/6 mice.
Ad libitum (AL)
AL + IGF-1
20% CR + IGF-1
30% CR + IGF-1
40% CR + IGF-1
1Means and standard errors obtained from univariate analyses, n=5 for all treatments.
2p and r2values obtained from Analysis of Variance.
Figure 1. Effects of calorie restriction and IGF-1 on body weight of
C57BL/6 mice. Error bars are one standard error.
from similar validation studies (22, 26). In the remainder of
the paper, untransformed DXA measurements are reported
for ease of comparison with past studies.
Growth, body composition and bone characteristics. Calorie
restriction resulted in significant changes in growth
characteristics (Figure 1). AL-fed animals gained weight
continuously, 20% and 30% CR animals maintained
approximately stable weights, and 40% CR animals showed
rapid weight loss followed by weight stabilization at about
25% lower weight than at study onset. Food consumption
data (not shown) indicated that CR-treated mice consumed
their entire ration. There were significant treatment effects
on all body composition and bone characteristics except
vertebral bone density (Table II). CR resulted in dose-
dependent decreases in body weight and fat weight (Table
II). CR also influenced lean weight and bone density, but
these responses were non-linear. Lean weights of animals
from both the 20% and 30% CR treatments were about 15%
lower than in the AL treatment and were 28% lower in the
40% CR group. Compared to AL-fed animals, overall bone
density was lower in CR animals, with the largest reduction
in bone density in the 30% CR group (Table II). We
contrasted CR and IGF-1 effects on total bone density with
bone density in the tibia and in vertebrae. Examination of
regional differences in rodent bone density is near the limits
of DXA resolution (26). Univariate analysis indicated
significant effects of treatments on tibial bone density (Table
II, p=0.007), but no effect on vertebral bone density (Table
II, p=0.173). Note that all three measurements of bone were
correlated (Total Bone Density vs. Tibia; r=0.74, p<0.001;
Total Bone Density vs. Vertebrae; r=0.47, p=0.002; Tibia vs.
Vertebrae; r=0.39, p=0.013).
We further analyzed the effects of CR and IGF-1
treatment by comparing CR versus AL-fed animals and
IGF-1-treated versus CR animals with and without
adjustment for body weight by analysis of covariance
(ANCOVA). In these tests, we pooled different treatments
to increase statistical power. Calorie restriction significantly
reduced bone mineral density (p<0.001), but this effect was
due entirely to the influence of CR on body weight; after
adjustment for body weight, the effect of CR on bone
mineral density was not significant (p=0.91). IGF-1
treatment increased bone mineral density whether or not
results were adjusted for body weight (p<0.001 unadjusted
for weight; p=0.002 with weight adjustment). This result is
also illustrated in Figure 2. Fat content was reduced by CR
with (p<0.001) and without (p<0.001) adjustment for
weight, but IGF-1 treatment did not influence fat content.
Serum IGF-1 and IGFBPs. After 26 days on treatment, total
serum IGF-1 concentrations were significantly reduced by
CR (p<0.0001), and there was a dose-response relationship
between the level of CR and the magnitude of this decrease
(Figure 3; p<0.001). Serum IGF-1 levels in AL-fed animals
averaged 785 ng/ml and were reduced to 616, 479 and 357
ng/ml in the 20, 30 and 40% CR treatment groups. IGF-1
treatment did not increase serum IGF-1, but instead
resulted in small decreases in serum IGF-1 levels (Figures
3 and 4A); this decrease averaged 26% for all four groups
receiving IGF-1 treatment and was significantly different
from zero (p<0.001). Total IGF-binding protein activity
in vivo 19: 667-674 (2005)
Figure 2. Effects of IGF-1 treatment on bone density of C57BL/6 mice.
The histograms represent differences in mean bone density of study groups
with and without IGF-1 treatment. Error bars are one standard error, and
asterisks denote significance levels, with *p<0.05; ** p<0.01. A significant
p value indicates that IGF-1 treatment increased bone density compared to
untreated animals at the same level of CR.
Figure 3. Effects of calorie restriction and IGF-1 treatment on serum
IGF-1 levels in C57BL/6 mice at study termination (26 days). AL-fed
mice, 20% CR and 30% CR mice received murine IGF-1 (20 Ìg/day) or
placebo; 40% CR mice received 80 Ìg/day murine IGF-1 or placebo.
Error bars are one standard error. Values with different superscripts are
significantly different (p<0.05).
(expressed as a percentage of the AL-fed + placebo pellet
group, mean ± S.E.) decreased in 30 and 40% CR-treated
animals (20% CR=106.2±6.8; 30% CR=85.7±10.1; 40%
CR=62.6±4.0). The effect of CR was significant (p=0.002,
but total IGF-binding was not influenced by IGF treatment
To further characterize serum levels of IGF-1 in response
to CR and implantation of time-release IGF-1 pellets, we
made repeated measures of IGF-1 over a period of 28 days
on mice from three treatment groups; AL fed, 30% CR and
30% CR + 20 Ìg/day IGF-1. CR reduced serum IGF-1
levels, and in this experiment IGF-1 treatment partially
restored these levels (Figure 4A). However, the increase in
serum IGF-1 compared to AL-fed controls was transient,
persisting for only about one week after pellet implantation.
AL-fed animals were larger than CR animals, and IGF-1-
treated CR animals were smaller than CR animals
(p<0.001; Figure 4B).
Some evidence suggests that calorie restriction (CR) or,
alternatively, targeting CR-related pathways, could contribute
to cancer prevention and control (4). One of the main
pathways through which CR acts to prevent cancer is the IGF-
1 pathway (4, 7, 27). CR animals have significant reductions
in serum concentrations of IGF-1 and are, therefore, thought
to have reduced signaling through the IGF-1 receptor, with
subsequent reduction in pro-cell survival pathways. Past
studies have shown that CR reduces body weight and fat
contents, and may influence bone characteristics (2). Our
results extend this work by characterizing the dose-response
relationship between CR and body composition/bone
characteristics. We also demonstrate experimentally that
reductions in bone density caused by CR can be reversed by
administration of IGF-1 and establish that time-release
capsules provide a minimally invasive approach to exploring
the physiological and molecular effects of IGF-1 on mice.
Overall, our results support the argument that efforts to use
CR or energy balance-related mechanistic targets to
ameliorate the effects of aging and/or to delay carcinogenesis
will require attention to potential effects of the IGF-1 pathway
on bone (4, 20, 27-30).
Increased body weight is associated with increased bone
density in humans and rodents (15, 31, 32). Furthermore,
reduction of bone density in rhesus monkeys caused by 30%
CR appears to be mediated through CR effects on body size
(30). Our study demonstrates that experimental reduction
in weight loss via CR also reduces bone mineral density, but
these effects are non-linear. For example, body weights of
40% CR animals were reduced by 38% compared to AL-fed
animals, but bone mineral density was reduced by only
9.3%. In contrast, the body weight of 30% CR animals was
reduced only 18.5% and bone density was reduced 10.4%.
Thus, the effects of CR on bone characteristics are non-
linear, and 40% CR has body weight-independent effects on
Treatment with IGF-1 increased bone density overall and
in the vertebrae, but not in the tibia. Effects of CR and
IGF-1 on bone characteristics are also dependent on bone
type and genetic differences between strains and species
(19). One study reports that IGF-1 treatment in growing
rats does not influence tibial bone density (33). Other
studies of mice suggest the effects of CR and IGF-1 on bone
characteristics are dependent on mouse strain (34-36). For
example, C57BL/6, DBA/2 and SENCAR mice differ in
their response to CR, and 40% CR in SENCAR mice
increases tibial bone density (35). Comparison of our
findings with those of mice congenitally deficient in IGF-1,
such as MIDI, LID and ALSKO (20, 28) may be
informative regarding the role of IGF-1 in determining bone
characteristics. MIDI mice have serum IGF-1 levels reduced
Berrigan et al: Calorie Restriction and IGF-1 Effects on Mouse Bone and Body Composition
Figure 4. Time course effects of CR and IGF-1 treatment on IGF-1 and
body weight. Error bars are one standard error. A. Serum IGF-1 levels in
AL-fed, 30% CR and 30% CR mice treated with IGF-1 via a time-release
pellet. B. Body weights.
by ~60% compared to those in wild-type controls, and LID
mice show a 75% reduction in IGF-1 levels. Similarly, CR
also reduces IGF-1 levels, with 40% CR resulting in an
almost 50% decrease in IGF-1 levels. Each of the
congenitally-deficient mouse strains also have significantly
reduced bone density, with changes in both trabecular and
cortical bone characteristics (20). Thus, not only are there
strain and species differences in bone responses to CR and
IGF-I, but the response of bone may differ based on
whether reductions in serum IGF-1 concentrations are
caused by CR-induced versus congenital perturbations.
Serum IGF-1 levels were significantly decreased by calorie
restriction. This is consistent with a number of past studies
(6, 7, 37). However, in the current study IGF-1 treatment
resulted in long-term decreases in serum IGF-1. This result
contrasts with other studies that report increased serum IGF-1
in mice receiving IGF-1 (7, 20). Previous studies have largely
used injection or osmotic mini-pumps for IGF-1 delivery.
Stress levels of mice in past studies could be increased due to
implantation of osmotic pumps or use of injections. However,
serum urinary corticosterone measurements output did not
differ between animals receiving pellets vs. osmotic pumps in
our study, nor did we observe elevated serum IGF-1 in
AL-fed mice receiving IGF-1 from pumps (data not shown).
Almost all past studies in rodent models have used human
IGF-1, but our results do not suggest that the use of murine
vs. human IGF-1 accounts for differences between this study
and others. Mice in our study that received human rather
than murine IGF-1 did not show elevated serum levels of
IGF-1 (data not shown). Treatment with IGF-1 did not
influence total IGF-binding protein activity in serum. Twice
daily injections, such as those used in some past studies (27),
could result in elevated serum levels of IGF-1 if serum is
collected shortly after injections, even if the kinetics of IGF-
binding proteins are changed by treatment. Further
experiments to explore these possibilities are underway.
Repeated measurements of IGF-1 levels in treated mice
indicated that serum IGF-1 did increase for the first week on
study (Figure 4A); measurements of IGF-1 receptor over
time could be informative.
Translation of discoveries concerning cancer prevention
via nutritional interventions requires renewed focus on costs
as well as benefits of candidate interventions. The major
focus of our research is the role of energy balance in cancer
prevention and control (4, 7, 21, 37), and we are particularly
concerned about the potential for deleterious side-effects on
bone characteristics that might be associated with preventive
interventions targeting energy balance-related pathways,
including the IGF-1 pathway. The present results indicate
that this pathway could mediate a tradeoff between the
beneficial results of CR in relation to cancer and negative
effects of weight loss on bone characteristics. In this paper,
we establish that treatment with murine recombinant IGF-1
delivered by time-release pellets reverses some of the effects
of calorie restriction on bone characteristics. We are now
exploring the effects of IGF treatment on gene expression.
Preliminary results suggest that IGF-1 treatment reverses
some of the gene expression changes induced by calorie
restriction (J. Lavigne, personal communication). Identifying
and characterizing the best systems for studying the
combined effects of CR on carcinogenesis and bone is an
ongoing challenge (38); C57BLl6 mice appear to be a useful
model for further studies of this topic.
D. Berrigan and J. Lavigne gratefully acknowledge the NCI’s
Cancer Prevention Fellowship Program for support while
carrying out this project. We thank Dr. Gregory Buzard for
performing the IGF Binding Protein Assays. We also
acknowledge NIH support via the Clinical Nutrition Research
Center Award (P30-DK56336) and the Center for Metabolic
Bone Disease (P30-AR46301), both at the University of Alabama
at Birmingham. This work was also supported in part with
federal funds from the National Cancer Institute under contract
N01-CO-12400 to SAIC-Frederick. All of us thank Dan Logsdon,
Chris Perella, Lisa Riffle, and the entire staff of the NCI-
Frederick animal facility for their conscientious help. Animal
care was provided in accordance with the procedures outlined in
the "Guide for the Care and Use of Laboratory Animals" (NIH
Publication No. 86-23, 1985). The content of this publication
does not necessarily reflect the views or policies of the
Department of Health and Human Services, nor does mention of
trade names, commercial products, or organizations imply
endorsement by the U.S. Government.
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Received April 6, 2005
Accepted April 18, 2005
Berrigan et al: Calorie Restriction and IGF-1 Effects on Mouse Bone and Body Composition