Am J Physiol Endocrinol Metab 307: E813–E821, 2014.
First published September 9, 2014; doi:10.1152/ajpendo.00256.2014.
Long-lived crowded-litter mice have an age-dependent increase in protein
synthesis to DNA synthesis ratio and mTORC1 substrate phosphorylation
Joshua C. Drake,1 Danielle R. Bruns,1 Frederick F. Peelor 3rd,1 Laurie M. Biela,1 Richard A. Miller,2
Karyn L. Hamilton,1* and Benjamin F. Miller1*
1
Health and Exercise Science Department, Colorado State University, Fort Collins, Colorado; and 2Department of Pathology
and Geriatrics Center, University of Michigan, Ann Arbor, Michigan
Submitted 5 June 2014; accepted in final form 4 September 2014
slowed aging; crowded litter; proteostasis; deuterium; mTORC1
in protein turnover
results in an accumulation of damaged proteins and propagates the development of the aging phenotype (36, 37). In
tissues that have a limited proliferative capacity, such as
heart and skeletal muscle, the ability to maintain functional
proteins (i.e., proteostasis) has become a key outcome in
aging research as a means to maintain homeostasis with age
(1, 2). Thus, interventions that maintain protein stability
could lead to slowed aging.
In proliferative tissues, growth is accomplished by cell
replication, which requires the duplication of DNA and of
cellular machinery through increased protein synthesis (reviewed in Ref. 16). In tissues with limited ability to proliferate
(e.g., skeletal muscle), growth is accomplished by increased
protein synthesis, which may be followed by recruitment of
A PROGRESSIVE AGE-DEPENDENT REDUCTION
* K. L. Hamilton and B. F. Miller were co-Principal Investigators.
Address for reprint requests and other correspondence: J. C. Drake, 220
Moby B Complex, Colorado State University, Fort Collins, CO 80523 (e-mail:
[email protected]).
http://www.ajpendo.org
new DNA from satellite cell division to maintain the myonuclear domain (44). Since enzymatic capacity for protein repair
is low within cells, damaged proteins must be removed and
subsequently replaced through the synthesis of new protein
(20, 26), a process that does not necessarily involve cell
division or DNA recruitment. Therefore, the simultaneous
assessment of DNA synthesis and protein synthesis could
provide context as to whether the synthesis of new proteins are
directed toward new cells or to maintaining existing cellular
structures, a phenomenon consistent with slowed aging (11, 22,
23). Our laboratory has shown that, in lifelong caloric restriction (CR) (22, 23) and chronically rapamycin-treated mice
(11), DNA synthesis is decreased. However, in long-lived
models compared with controls, there are variable responses in
protein synthesis based on the protein fraction, tissue, model,
and sex. To date, no study, in a model of slowed aging or other,
has considered protein synthetic rates in the context of DNA
synthesis.
The mechanistic (formerly mammalian) target of rapamycin
(mTOR) signaling pathway promotes growth by increasing
protein synthesis and cellular cycling via two multiprotein
complexes, mTORC1 and mTORC2 (18). CR (17, 46) and
chronic rapamycin administration (14, 30, 45) suppress
mTORC1 activity and increase lifespan and multiple indexes
of health in various species. Although it has been proposed that
a decrease in mTOR signaling is a key mechanism of slowed
aging (18), it is unclear whether decreased mTOR signaling is
necessary for slowed aging.
The nutritional environment of the developing fetus can
have a profound impact on gene expression throughout the
lifespan of the offspring, affecting health and longevity (8, 13,
40). Furthermore, changing litter size or postnatal maternal
protein intake can change offspring lifespan, suggesting that
some degree of genomic plasticity also remains during the
postnatal suckling period (8, 12, 19, 38, 39). For example,
increasing litter size by 50% for the first 3 wk of life, a model
termed crowded litter (CL), extends mean and maximal lifespan (39). The increase in litter size presumably imposes a CR
period until the pups are weaned and given free access to food
(33, 39). How a transient nutrient stress at such a young age
extends lifespan is unknown. We hypothesized that, like other
long-lived models previously investigated in our laboratory
(11, 22, 23), CL mice would have decreased DNA synthesis
and mTORC1 signaling but maintained protein synthesis. In
addition, we hypothesized that if new protein synthesis was
considered in relation to new DNA synthesis, there would be
an increase in the ratio within the CL model, indicative of
greater preservation of existing protein structures.
0193-1849/14 Copyright © 2014 the American Physiological Society
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Drake JC, Bruns DR, Peelor FF 3rd, Biela LM, Miller RA,
Hamilton KL, Miller BF. Long-lived crowded-litter mice have an
age-dependent increase in protein synthesis to DNA synthesis ratio
and mTORC1 substrate phosphorylation. Am J Physiol Endocrinol
Metab 307: E813–E821, 2014. First published September 9, 2014;
doi:10.1152/ajpendo.00256.2014.—Increasing mouse litter size
[crowded litter (CL)] presumably imposes a transient nutrient stress
during suckling and extends lifespan through unknown mechanisms.
Chronic calorically restricted and rapamycin-treated mice have decreased DNA synthesis and mTOR complex 1 (mTORC1) signaling
but maintained protein synthesis, suggesting maintenance of existing
cellular structures. We hypothesized that CL would exhibit similar
synthetic and signaling responses to other long-lived models and, by
comparing synthesis of new protein to new DNA, that insight may be
gained into the potential preservation of existing cellular structures in
the CL model. Protein and DNA synthesis was assessed in gastroc
complex, heart, and liver of 4- and 7-mo CL mice. We also examined
mTORC1 signaling in 3- and 7-mo aged animals. Compared with
controls, 4-mo CL had greater DNA synthesis in gastroc complex with
no differences in protein synthesis or mTORC1 substrate phosphorylation across tissues. Seven-month CL had less DNA synthesis than
controls in heart and greater protein synthesis and mTORC1 substrate
phosphorylation across tissues. The increased new protein-to-new
DNA synthesis ratio suggests that new proteins are synthesized more
so in existing cells at 7 mo, differing from 4 mo, in CL vs. controls.
We propose that, in CL, protein synthesis shifts from being directed
toward new cells (4 mo) to maintenance of existing cellular structures
(7 mo), independently of decreased mTORC1.
INCREASED PROTEIN-TO-DNA SYNTHESIS RATIO AND MTOR IN CL MICE
METHODS
Animals and Treatments
Sample Preparation and Analysis of Analytes Via GC-MS
Protein. Protein was hydrolyzed by incubation for 24 h at 120°C in
6 N HCl. The hydrolysates were ion exchanged, dried under vacuum,
and resuspended in 1 ml of molecular biology grade H2O. Suspended
samples (500 ␮l) were derivatized [500 ␮l acetonitrile, 50 ␮l 1 M
K2HPO4 (pH 11), and 20 ␮l pentafluorobenzyl bromide (Pierce
Scientific, Rockford, IL)], sealed, and incubated at 100°C for 1 h.
Derivatives were extracted into ethyl acetate. The organic layer was
removed and dried by N2 followed by vacuum centrifugation. Samples were reconstituted in 1 ml of ethyl acetate and then analyzed.
The pentafluorobenzyl-N,N-di(pentafluorobenzyl) derivative of alanine was analyzed on an Agilent 7890A GC coupled to an Agilent
5975C MS, as previously described (11, 22, 23, 32). The newly
synthesized fraction (f) of proteins was calculated from the true
precursor enrichment (p) using plasma analyzed for 2H2O enrichment
and adjusted using mass isotopomer distribution analysis (MIDA) (5).
Protein synthesis was calculated as the ratio of 2H2O-labeled to
unlabeled alanine (5) bound in proteins over the entire labeling period
and expressed as fraction new in 2 wk.
Body water. To determine body water enrichment, 125 ␮l of
plasma was placed into the inner well of an o-ring screw-on cap and
placed inverted on a heating block overnight. Two microliters of 10 M
NaOH and 20 ␮l of acetone was added to all samples and to 20 ␮l
0 –20% 2H2O standards and capped immediately. Samples were vortexed at low speed and left at room temperature overnight. Extraction
was performed by the addition of 200 ␮l of hexane. The organic layer
was transferred through anhydrous Na2SO4 into GC vials and analyzed via EI mode using a DB-17MS column.
DNA. Determination of 2H incorporation into purine deoxyribose
(dR) of DNA from whole tissue and bone marrow was performed as
described previously (5, 11, 22, 32). Briefly, isolated DNA was
hydrolyzed overnight at 37°C with nuclease S1 and potato acid
Body Weight Comparison
Protein Isolation
Body Weight (g)
p = 0.179
30
p = 0.027
Whole tissue DNA synthesis in skeletal muscle, heart, and liver
was assessed according to procedures previously described (5, 11, 22,
23, 28). Approximately 75 ng/␮l (heart and gastroc complex) and
400ng/␮l (liver) of total DNA was extracted from ⬃15 mg tissue
(QiAamp DNA mini kit Qiagen, Valencia, CA, USA). DNA for the
*
20
Con
10
CL
m
L
C
on
7
7
m
C
C
L
4
4
o
m
o
o
o
m
n
on
-W
ea
C
L
Po
Po
st
st
-W
ea
n
0
C
DNA Isolation
p = 0.387
40
on
We assessed protein synthesis in three subcellular fractions: mitochondrial enriched (Mito), cytosolic (Cyto), and mixed (Mix). Mix
contains nuclei, plasma membranes, and, in the case of skeletal
muscle and heart, contractile proteins. Cyto contains all other intracellular components with the exception of mitochondria, which is
contained in the Mito fraction. Tissues were fractionated according to
our previously published procedures (11, 22, 23, 31). Briefly, tissues
(25– 60 mg) were homogenized 1:10 in isolation buffer (100 mM KCl,
40 mM Tris·HCl, 10 mM Tris base, 5 mM MgCl2, 1 mM EDTA, 1
mM ATP, pH 7.5) with phosphatase and protease inhibitors (HALT,
Thermo Scientific, Rockford, IL) using a bead homogenizer (Next
Advance, Averill Park, NY). After homogenization, subcellular fractions were isolated via differential centrifugation as previously described (11, 22, 23). Once fraction pellets were isolated and purified,
250 ␮l 1 M NaOH was added, and pellets were incubated for 15 min
at 50°C and 900 rpm.
Fig. 1. Body weight comparison of crowded-litter (CL) and control (Con) mice
postweaning, at 4 mo and at 7 mo of age. Postweaning CL mice weighed
significantly less than normal-litter-size Con mice. There was no difference in
body weight between CL and Con mice at 4 or 7 mo of age; n ⫽ 8 per group
for postweaning, n ⫽ 4 per group in 4-w and 7-mo-old cohorts. *P ⬍ 0.05 for
CL vs. Con.
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All procedures and conditions at the animal care facility meet or
exceed the standards for animal housing as described in the Animal
Welfare Act regulations and the Guide for the Care and Use of
Laboratory Animals and were approved by the University Committee
on Use and Care of Animals at the University of Michigan. We used
the genetically heterogeneous offspring of CB6F1 female and
C3D2F1 male (UM-HET3) mice, which have been well characterized
for the development of the CL model (38, 39).
Routine veterinary care was provided by the Biomedical Science
Research Building staff. UM-HET3 litters were culled to eight, and an
additional four mice from separate litters were added, resulting in a
50% increase in litter size (39). Control (Con) UM-HET3 mice were
maintained in a litter size of eight pups. After the 3-wk suckling
period, female mice were weaned onto a chow diet, and CL and Con
were placed in their own respective cages (4 mice per group per cage)
with ad libitum access to food and water. At 4 mo (not weight stable,
still growing) and 7 mo of age [weight stabilizing, and same age as our
previous investigations in long-lived models (11, 22, 39)], CL (n ⫽ 4,
per age group) and Con (n ⫽ 4, per age group) mice received
deuterium-labeled water (2H2O) for 2 wk. Animals received an
intraperitoneal injection of 99% enriched 2H2O to enrich the body
water pool (assumed 60% of body wt) to 8% (22, 28). Animals then
received 8% 2H2O in their drinking water with ad libitum access for
the next 2 wk. After the labeling period and following an overnight
fast (7 mo only), mice were euthanized using a CO2 overdose
according to the AVMA Guidelines on Euthanasia. Complete loss of
pedal reflexes was confirmed before tissues were collected. The
posterior aspect of the distal hindlimbs [gastroc complex: gastrocnemius, soleus, and plantaris (i.e., mixed skeletal muscle)], heart, liver,
bone marrow from the tibia, via flushing the cavity with PBS, and
plasma, via blood from cardiac puncture, were taken and immediately
frozen in liquid nitrogen for later analysis.
The 4-mo mice were not fasted the night before being euthanized.
Therefore, to assess mTORC1 signaling at a younger age, an additional cohort of ⬃3-mo CL and Con (n ⫽ 4, respectively) mice were
euthanized following an overnight fast, and gastroc complex and liver
were harvested as already reported.
precursor pool was obtained from bone marrow. DNA from bone
marrow was isolated by extracting ⬃300 mg from the tibial bone
marrow suspension which was centrifuged for 10 min at 2,000 g.
DNA from the resulting bone marrow pellet was extracted the same as
tissue samples already described.
C
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INCREASED PROTEIN-TO-DNA SYNTHESIS RATIO AND MTOR IN CL MICE
phosphatase. Hydrolysates were reacted with pentafluorobenzyl hydroxylamine and acetic acid and then acetylated with acetic anhydride
and 1-methylimidazole. Dichloromethane extracts were dried, resuspended in ethyl acetate, and analyzed by GC-MS as previously
described (11, 22, 32). The fraction new in 2 wk was calculated by
comparison with bone marrow (representing an essentially fully
turned over cell population and thus the precursor enrichment) in the
same animal (11, 22, 23).
New Protein-to-New DNA Synthesis Ratio
From the synthesis rates of protein and DNA, we calculated the
new protein-to-new DNA synthesis ratio. The rationale is that this
ratio illustrates how much protein is made in relation to the rate of
new DNA synthesis during the labeling period, giving insight into
whether new proteins are made in either existing or new cells.
A portion of the Cyto fraction from 7-mo and the additional
3-mo-fasted cohort was used for Western blot analysis. Protein con-
0.75
0.50
0.25
0.00
Fraction new Ala in 2 weeks
Cyto
Mito
4 mo Heart
D
Cyto
Fraction new Ala in 2 weeks
Mix
1.00
0.75
0.50
0.25
0.00
Mix
E
Mito
4 mo Liver
F
Cyto
1.5
1.0
0.5
0.0
Mix
Fraction new Ala in 2 weeks
1.00
C
Fraction new Ala in 2 weeks
B
4 mo Gastroc Complex
Fraction new Ala in 2 weeks
Fraction new Ala in 2 weeks
A
Mito
centration was determined using a bicinchoninic acid assay (Thermo
Fisher, Rockford, IL). Samples were diluted to the same concentration, boiled with Laemmli buffer, and then 30 – 45 ␮g of protein was
separated using 10% SDS-PAGE at 100 V. Proteins were transferred
at 4°C (100 V for 75 min in 20% wt/vol methanol, 0.02% wt/vol SDS,
25 mM Tris base, 192 mM glycine, pH 8.3) to nitrocellulose paper and
incubated in 5% nonfat dry milk in Tris-buffered saline with Tween
20 (TBST) for 1 h. Antibodies were purchased from Cell Signaling
Technologies [Boston, MA; rpS6 phospho-Ser235/236 no. 4858, rpS6
total no. 2217, eukaryotic initiation factor 4E binding protein-1
(4E-BP1) phospho-Thr37/46 no. 9459, 4E-BP1 total no. 9452], or
␤-tubulin no. sc-5274 (Santa Cruz Biotechnology, Santa Cruz, CA).
Blots were incubated overnight with primary antibodies diluted 1:500
(skeletal muscle rpS6), 1:1,000 (all others), 1:2,000 (liver rpS6), or
1:500 (␤-tubulin). Blots were washed in TBST and incubated with
anti-rabbit or anti-mouse (␤-tubulin) HRP-conjugated secondary antibodies diluted 1:5,000 in 5% milk with subsequent chemiluminescence detection (West Dura; Pierce, Rockford, IL). Images were
captured and densitometry was analyzed by UVP Bioimaging system
7 mo Gastroc Complex
1.00
0.75
0.50
0.25
*
*
*
Cyto
Mito
0.00
Mix
7 mo Heart
1.00
0.75
0.50
*
*
Mix
Cyto
*
0.25
0.00
Mito
7 mo Liver
1.00
*
*
*
Cyto
Mito
Fig. 2. Protein synthesis in gastroc complex (gastrocnemius, soleus, and plantaris, i.e., mixed skeletal muscle), heart, and liver over 2 wk in 4-mo and
7-mo CL and Con. There were no differences in
protein synthesis between groups in any tissue
between 4-mo CL and 4-mo Con (A, C, and E).
Protein synthesis was significantly higher in all
tissues and fractions in 7-mo CL vs. 7-mo Con (B,
D, and F); n ⫽ 4 per group and fraction except Con
liver Mix n ⫽ 3 because of an unquantifiable peak
on the GC. Two values in liver 7-mo CL Cyto and
Mito were fully turned over (i.e., 100% new) after
2 wk, and so calculations were performed with a
synthesis value of 1.00 for those animals. *P ⬍
0.05 for CL vs. Con; no. P ⬍ 0.05 difference
between pooled (Con and CL) fractions in all tissues at both 4 and 7 mo.
0.75
0.50
0.25
0.00
Mix
# significant difference between sub-cellular fractions independent of treatment
Con
CL
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Western Blotting
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E816
INCREASED PROTEIN-TO-DNA SYNTHESIS RATIO AND MTOR IN CL MICE
(Upland, CA). Blots were probed for phosphorylated proteins first,
placed in stripping buffer (GM Biosciences, Rockville, MD), and then
reprobed for total proteins. Equal loading was verified using Ponceau
S staining and ␤-tubulin. Due to undetectable rpS6 in skeletal muscle
homogenate, a portion of the homogenate was acetone precipitated
and then analyzed via Western blotting. Phosphorylated proteins were
expressed as a ratio with total.
Statistics
RESULTS
Protein and DNA Synthesis
Body weight at weaning was significantly reduced in CL
compared with Con mice (Fig. 1), suggesting that CL mice
were calorically restricted during suckling (33, 39). However,
there were no differences in body weight at either 4 or 7 mo of
age (Fig. 1). In the gastroc complex, heart, and liver of 4-mo
CL mice, there were no differences in protein synthesis within
the Mix, Cyto, or Mito fractions compared with 4-mo Con
(Fig. 2, A, C, and E). By 7 mo of age, there was a dramatic
change, in the gastroc complex, heart, and liver (Fig. 2, B, D,
and F) all having significantly greater rates of protein synthesis
in Mix, Cyto, and Mito fractions compared with 7-mo Con
(Fig. 2, B, D, and F). Independent of treatment, protein synthesis was significantly different between subcellular fractions
in all tissues and ages (Fig. 2, A–F).
DNA synthesis was significantly greater in the gastroc complex of 4-mo CL mice compared with Con (Fig. 2A). DNA
synthesis in heart trended greater in 4-mo CL compared with
4-mo Con but did not reach statistical significance (P ⫽ 0.070;
Fig. 3A). Liver DNA synthesis in 4-mo CL was not different
from that of 4-mo Con (Fig. 3A). At 7 mo, CL mice had
0.15
0.05
p=0.070
*
0.00
Gastroc
Complex
Heart
mTORC1 Signaling
At 3 mo (additional fasted cohort), there were no differences
between rpS6 and 4E-BP1 phosphorylation between groups in
either gastroc complex or liver (Fig. 5). At 7 mo, phosphorylation of rpS6 was significantly increased in heart and liver
(Fig. 6, B and C). Phosphorylation of 4E-BP1 was significantly
increased in gastroc complex (Fig. 6D). Although it did not
reach statistical significance, 4E-BP1 in heart (Fig. 4E) was
P ⫽ 0.055.
DISCUSSION
Overview of Primary Findings
We tested the hypothesis that, compared with Con, CL mice
would have decreased DNA synthesis and mTORC1 signaling,
and maintained protein synthesis rates. Furthermore, we hypothesized that when the synthesis of new proteins was compared with the synthesis of new DNA there would be changes
in the CL model that indicated a greater preservation of
existing cellular structures. At 4 mo of age, protein synthesis
was not different between CL and Con in any tissue but DNA
synthesis was significantly increased in gastroc complex and
trended in the heart of CL compared with Con. At 7 mo of age,
however, protein synthesis rates were significantly greater in
all tissues and fractions, whereas DNA synthesis was signifi-
B
4 mo DNA
0.10
In the 4-mo CL, the new protein/new DNA synthesis ratio
was significantly decreased compared with 4-mo Con across
subcellular fractions in both the gastroc complex and heart
(Fig. 4, A and C). At 4 mo, in the liver there was no difference
between Con and CL in the new protein/new DNA synthesis
ratio (Fig. 4E). However, at 7 mo, the new protein/new DNA
synthesis ratio was significantly increased across subcellular
fractions in gastroc complex, heart, and liver (Fig. 4, B, D,
and F).
Liver
Fraction new dR in 2 weeks
Fraction new dR in 2 weeks
A
New Protein/New DNA Synthesis Ratio
7 mo DNA
0.15
Con
CL
0.10
*
0.05
0.00
Gastroc
Complex
Heart
Liver
Fig. 3. DNA synthesis in gastroc complex, heart, and liver over 2 wk in 4-mo and 7-mo CL and Con. A: gastroc complex DNA synthesis was significantly greater
in 4-mo CL than in 4-mo Con, heart DNA synthesis in 4-mo CL tended to be greater (P ⫽ 0.070) than 4-mo Con, and there were no differences in liver DNA
synthesis between 4-mo animals. B: in 7-mo animals, DNA synthesis was significantly less in CL heart than in Con, and no significant differences were observed
between 7-mo CL and 7-mo Con in gastroc complex or liver. DNA synthesis rates in heart tissues from 4-mo mice were log transformed to normalize variance;
n ⫽ 4 in both ages and groups except that in 7-mo Con gastroc complex and 7-mo CL gastroc complex and heart n ⫽ 3 due to one data point being excluded
as an outlier. *P ⬍ 0.05 for CL vs. Con.
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Statistical analysis was performed using Prism v.4.0c (GraphPad
Software, La Jolla, CA). Comparisons between treatment groups and
protein synthesis and new protein/new DNA synthesis ratio data in the
various subcellular fractions were assessed by two-way ANOVA.
DNA synthesis and Western blot data were assessed via two-sided
Student’s t-test. Variances were normalized via log transformation
where necessary. Significance was set at P ⬍ 0.05, and P values of
ⱕ0.10 are noted. Data are presented as means ⫾ SE.
significantly lower rates of DNA synthesis in the heart but not
in gastroc complex and liver (Fig. 3).
INCREASED PROTEIN-TO-DNA SYNTHESIS RATIO AND MTOR IN CL MICE
E817
cantly less in the heart of CL than in Con. By comparing the
synthesis of new protein to the synthesis of new DNA, we
present novel insight into the potential destination of new
proteins in long-lived CL mice. We demonstrate for the first
time that, compared with normal litter sized controls, the
transient nutrient stress of a 50% increase in litter size leads to
alterations in the new protein/new DNA ratio later in life,
suggesting a switch in which fewer of the new proteins synthesized are directed toward new cells and more are directed
toward the maintenance of existing cellular structures. Finally,
we report the novel finding of tissue-dependent increases in
mTORC1 activity at 7 mo compared with no difference at 3 mo
in the long-lived CL mice, suggesting that a decrease in
mTORC1 may not be requisite for lifespan extension in CL.
Postnatal Feeding and Growth
Gene expression during in utero development can be profoundly affected by maternal nutrition, having lifelong effects
on the healthspan and longevity of offspring (8, 13, 40).
Changes in lifespan and healthspan through litter reduction in
rats (3– 4 pups/litter) (12) as well as litter enlargement (i.e.,
CL) experiments in mice (38, 39) suggest that nutritional status
during early development can also have long-lasting effects.
For example, reducing litter size increases pups’ caloric intake,
leading to a vast array of pathologies associated with poor
health (12). Alternatively, CL extends mean and maximal
lifespan (39) and upregulates the transcription of some phase I
xenobiotic enzymes (38), which are protective against environmental toxins, and improves multiple markers of metabolic
health (33). However, it is not known how an increase in litter
size affects the synthetic processes that contribute to growth
and increased lifespan and healthspan.
The use of 2H2O to measure DNA synthesis is reflective of
S-phase synthesis, not DNA repair or random incorporation (6,
28). In our previous studies using models of slowed aging (11,
22, 23) and human exercise (32), we repeatedly demonstrated
synthesis of new DNA in tissues comprised predominantly of
postmitotic cells (i.e., heart and skeletal muscle). In the present
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Fig. 4. New protein-to-new DNA synthesis ratios in
gastroc complex and heart of CL and Con. At 4 mo,
there was a significant decrease in the new protein/
new DNA ratio in CL compared with Con in Mix,
Cyto, and Mito fractions in both gastroc complex
and heart (A and C) but no change in any fraction
between groups in liver (E). At 7 mo, there was a
significant increase in the new protein/new DNA
ratio in Mix, Cyto, and Mito fractions in gastroc
complex, heart, and liver of CL compared with Con
(B, D, and F); n ⫽ 4 per group, except 7-mo gastroc
complex Con and CL and 7-mo heart CL n ⫽ 3. See
legends for Figs. 1 and 2. *P ⬍ 0.05 for CL vs. Con.
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INCREASED PROTEIN-TO-DNA SYNTHESIS RATIO AND MTOR IN CL MICE
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Fig. 5. Western blotting of mTOR complex 1
(mTORC1) substrates rpS6 and 4E-BP1 in fasted
3-mo CL mice. There were no differences between
3-mo CL and Con in rpS6 or 4E-BP1 phosphorylation in gastroc complex (A and C) or liver (B and D).
Data are expressed as a ratio of phosphorylated to
total protein. ␤-Tubulin is shown to verify equal
loading of protein. In gastroc complex rpS6, CL n ⫽
3 because one band was not visible to assess and
thus was excluded to avoid misrepresenting the
data; n ⫽ 4 in all other groups.
study, we found changes in DNA synthesis within tissues
comprised predominantly of postmitotic cells that is consistent
with our previous investigations (11, 22, 23, 32). Therefore, we
propose two possibilities for measurable changes in DNA
synthesis in postmitotic tissues: 1) there is some mitotic ability
in cardiac myocytes and skeletal muscle myofibers that are not
fully appreciated; or 2) there are other cellular sources of new
DNA in these tissues that change based on our interventions.
For example, cardiomyocytes have some ability to replicate
their own DNA (4, 35) or incorporate DNA from other neighboring cell types (3, 29). Furthermore, regulation of cardiac
DNA synthesis may vary by stage of development (42), which
is supportive of the varying age-dependent DNA synthesis
rates in CL heart. Similarly, skeletal muscle is primarily made
up of postmitotic, multinucleated myofibers but also contains
resident proliferative satellite cells, pericytes, interstitial cells,
and myoendothelial cells (10, 25, 47). When myofiber size
increases, requiring an increase in protein synthesis, there is an
increase in satellite cell recruitment (i.e., new DNA) to stabilize the myonuclear domain (32, 41), which isotopic labeling
reflects (27). Previous work in exercising humans from our
laboratory suggest that it is unlikely that mitochondrial DNA
synthesis contributes significantly to our measurement of total
DNA synthesis in skeletal muscle using 2H2O (32), although
there could be a species difference in mice. Therefore, the
increase in DNA synthesis in skeletal muscle of 4-mo CL may
indicate a period of catch-up growth. Importantly, we assessed
DNA synthesis by extracting DNA from whole tissue. Thus,
the age- and tissue-dependent changes we present here are
likely the summation of the various cell types within multiple,
primarily postmitotic, tissues of the long-lived CL mouse
model. Collectively, our current data in CL mice provide novel
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INCREASED PROTEIN-TO-DNA SYNTHESIS RATIO AND MTOR IN CL MICE
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insights into growth regulation across whole tissue at different
ages.
Insights from Simultaneous Assessment of Protein and DNA
Synthesis
Low enzymatic capacity for protein repair within cells
means that damaged proteins must be removed and replaced
through synthesis of new proteins (20, 26). In light of our
previous investigations in long-lived models, we posit that
maintaining rates of protein synthesis while DNA synthesis is
decreased suggests that newly synthesized proteins are made
primarily in existing cells, which may promote the maintenance of existing cellular structures (i.e., proteostasis), contributing to increased lifespan and healthspan (2, 11, 22, 23). In
contrast to our investigations into long-lived CR and chronic
rapamycin-fed mouse models of the same age (11, 22), the
7-mo long-lived CL mice had increased protein synthesis in all
tissues and subcellular fractions in gastroc complex, heart, and
liver. It is important to note that the CL model is generated
from mice of the same heterogeneous background (UM-HET3)
as in our previous investigation of chronic rapamycin feeding
(11); thus, strain differences cannot account for their differing
response in protein synthesis. The elevated rates of mitochondrial protein synthesis in 7-mo CL, indicative of mitochondrial
biogenesis (21), is consistent with our previous investigations
and hypothesized to be key to slowed aging (11, 22, 24).
Therefore, determining whether the increase in protein synthesis in 7-mo CL was occurring in new cells or existing cells may
provide insight into mechanism(s) responsible for increased
lifespan in CL.
To gain insight into what proportion of newly synthesized
proteins are going to new cells vs. existing cellular structures,
AJP-Endocrinol Metab • doi:10.1152/ajpendo.00256.2014 • www.ajpendo.org
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Fig. 6. A–F: Western blotting of mTORC1 substrates rpS6 and 4E-BP1 in 7-mo CL mice. rpS6 phosphorylation was significantly greater in heart and liver in
7-mo CL than in Con (B and C). 4E-BP1 phosphorylation was significantly greater in gastroc complex and tended to be greater in heart (P ⫽ 0.055) in 7-mo
CL vs. 7-mo Con (D and E). Data are expressed as ratio of phosphorylated to total protein. ␤-Tubulin is shown to verify equal loading of protein. Liver rpS6
was log transformed to normalize variance; n ⫽ 4 per group. *P ⬍ 0.05 for CL vs. Con.
E820
INCREASED PROTEIN-TO-DNA SYNTHESIS RATIO AND MTOR IN CL MICE
we compared the synthesis of new protein to the respective
synthesis of new DNA. Comparing 4-mo CL to 4-mo Con, it
appears that a greater proportion of protein synthesis is directed
to cellular expansion. In contrast, in 7-mo CL mice compared
with 7-mo Con, new proteins are synthesized primarily in
existing cells, as illustrated by a significant increase in new
proteins synthesized in relation to new DNA, indicative of new
cells added to the population, across tissues. In summary, we
speculate that in CL mice a tradeoff from growth to proteostasis occurs between 4 mo and 7 mo of age that is characterized
by an increase in the synthesis of new protein/new DNA ratio
across subcellular fractions. Therefore, maintained proteostasis
may be a critical determinant in the extended lifespan and
healthspan of CL mice.
the lifespan. Collectively, we expand on the body of evidence
establishing CL as a novel model of longevity that provides
valuable insight into protein synthesis and its role in lifespan
and healthspan that could aid in the development of novel
strategies to promote slowed aging.
ACKNOWLEDGMENTS
We thank Kathryn Baeverstad for assistance with Western blotting and
Sabrina Van Roekel for technical assistance.
GRANTS
This project was funded by National Institutes of Health grants 1K01
AG-O31829-01 to B. F. Miller, NIH R01 AG-042569 to B. F. Miller and K. L.
Hamilton, and R01 AG-019899 to R. A. Miller.
No conflicts of interest, financial or otherwise, are declared by the author(s).
mTORC1 is a central regulator of protein turnover, cell
cycle, and mRNA translation (18). Inhibiting or downregulating mTORC1 signaling is suggested to be integral to the
lifespan extension imparted by CR and chronic rapamycin
treatment (14, 15, 22, 23). Expanding upon our protein synthesis findings in long-lived CL mice, we assessed two
mTORC1 substrates, rpS6 and 4E-BP1, which are involved in
rRNA transcription and translation initiation, respectively (7,
43). In young fasted (⬃3 mo of age) mice, phosphorylation of
rpS6 and 4E-BP1 was not different between CL and Con mice
in any tissue, corroborating the 2-wk protein synthesis measurements in 4-mo CL. In contrast, 7-mo CL mice had greater
rpS6 phosphorylation in heart and liver, whereas 4E-BP1
phosphorylation was greater in the gastroc complex compared
with 7-mo Con, consistent with the increased rates of protein
synthesis observed over 2 wk in CL at 7 mo. An increase in
phosphorylation of mTORC1 substrates is in contrast to the
reduced mTORC1 activity seen in age-matched calorie-restricted, rapamycin-treated (11, 22), and S6K1⫺/⫺ mice (34) as
well as the unaltered mTORC1 activity in methionine-restricted mice (39). However, branched-chain amino acid
(BCAA) supplementation extends lifespan while subsequently
increasing mTORC1 activity (9). Together with our current
data in the CL model, this suggests that although decreasing
mTORC1 activity is sufficient to extend lifespan, it may not be
necessary. We propose that increases in proteostatic mechanisms may be more important factors in lifespan and healthspan than decreased mTORC1 (39).
Summary and Conclusion
Here, we demonstrate in long-lived CL mice that a transient
nutrient stress during the suckling period results in long-term
alterations in protein and DNA synthesis that differ between 4
and 7 mo. We propose that, compared with normal-litter-size
controls, there is a greater proportion of protein synthesis
directed to new cells at 4 mo, but it switches to a greater
proportion of protein synthesis in existing cells, thus being
directed to the maintenance of existing cellular structures, at 7
mo. We also present novel data indicating that, in CL mice,
lifespan extension may be accomplished independently of
reduced mTORC1 activity in primarily postmitotic tissues.
These data highlight the importance of assessing pathways
thought to be integral to slowed aging, such as mTORC1, as
well as the metabolic outcomes, at multiple time points across
AUTHOR CONTRIBUTIONS
Author contributions: J.C.D., R.A.M., K.L.H., and B.F.M. conception and
design of research; J.C.D., D.R.B., F.F.P., and L.M.B. performed experiments;
J.C.D., D.R.B., F.F.P., and B.F.M. analyzed data; J.C.D., D.R.B., K.L.H., and
B.F.M. interpreted results of experiments; J.C.D. prepared figures; J.C.D.
drafted manuscript; J.C.D., D.R.B., R.A.M., K.L.H., and B.F.M. edited and
revised manuscript; J.C.D., D.R.B., F.F.P., L.M.B., K.L.H., and B.F.M. approved final version of manuscript.
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