Article

An aging Interventions Testing Program: Study design and interim report

Department of Pathology and Geriatrics Center, University of Michigan, Ann Arbor VA Medical Center, Ann Arbor, MI 48109-2200, USA.
Aging Cell (Impact Factor: 6.34). 09/2007; 6(4):565-75. DOI: 10.1111/j.1474-9726.2007.00311.x
Source: PubMed
ABSTRACT
The National Institute on Aging's Interventions Testing Program (ITP) has developed a plan to evaluate agents that are considered plausible candidates for delaying rates of aging. Key features include: (i) use of genetically heterogeneous mice (a standardized four-way cross), (ii) replication at three test sites (the Jackson Laboratory, TJL; University of Michigan, UM; and University of Texas, UT), (iii) sufficient statistical power to detect 10% changes in lifespan, (iv) tests for age-dependent changes in T cell subsets and physical activity, and (v) an annual solicitation for collaborators who wish to suggest new interventions for evaluation. Mice in the first cohort were exposed to one of four agents: aspirin, nitroflurbiprofen (NFP), 4-OH-alpha-phenyl-N-tert-butyl nitrone (4-OH-PBN), or nordihydroguiaretic acid (NDGA). An interim analysis was conducted using survival data available on the date at which at least 50% of the male control mice had died at each test site. Survival of control males was significantly higher, at the interim time-point, at UM than at UT or TJL; all three sites had similar survival of control females. Males in the NDGA group had significantly improved survival (P = 0.0004), with significant effects noted at TJL (P < 0.01) and UT (P < 0.04). None of the other agents altered survival, although there was a suggestion (P = 0.07) of a beneficial effect of aspirin in males. More data will be needed to determine if any of these compounds can extend maximal lifespan, but the current data show that NDGA reduces early life mortality risks in genetically heterogeneous mice at multiple test sites.

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Available from: Martin A Javors
Aging Cell
(2007)
6
, pp565–575 Doi: 10.1111/j.1474-9726.2007.00311.x
© 2007 The Authors
565
Journal compilation © Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2007
Blackwell Publishing Ltd
An aging Interventions Testing Program: study design
and interim report
Richard A. Miller,
1
David E. Harrison,
2
Clinton M. Astle,
2
Robert A. Floyd,
3
Kevin Flurkey,
2
Kenneth L. Hensley,
3
Martin A. Javors,
4
Christiaan Leeuwenburgh,
5
James F. Nelson,
6
Ennio Ongini,
7
Nancy L. Nadon,
8
Huber R. Warner
8
and Randy Strong
9
1
Department of Pathology and Geriatrics Center, University of
Michigan, Ann Arbor VA Medical Center, Ann Arbor,
MI 48109-2200, USA
2
The Jackson Laboratory, Bar Harbor, ME 04609, USA
3
Oklahoma Medical Research Foundation, Oklahoma City,
OK 73104, USA
4
Department of Psychiatry, University of Texas Health Science
Center at San Antonio, TX 78229, USA
5
Department of Aging and Geriatrics, University of Florida,
Gainesville, FL 32610, USA
6
Department of Physiology and Barshop Center for Longevity and
Aging Studies, University of Texas Health Science Center,
San Antonio, TX 78229, USA
7
NicOx Research Institute, Milan, Italy
8
Biology of Aging Program, National Institute on Aging, Bethesda,
MD 20892, USA
9
Geriatric Research, Education and Clinical Center and Research
Service, South Texas Veterans Health Care System, San Antonio; and
Department of Pharmacology, and Barshop Center for Longevity
and Aging Studies at The University of Texas Health Science Center
at San Antonio, TX 78229, USA
Summary
The National Institute on Aging’s Interventions Testing
Program (ITP) has developed a plan to evaluate agents
that are considered plausible candidates for delaying
rates of aging. Key features include: (i) use of genetically
heterogeneous mice (a standardized four-way cross),
(ii) replication at three test sites (the Jackson Laboratory, TJL;
University of Michigan, UM; and University of Texas, UT),
(iii) sufficient statistical power to detect 10% changes in
lifespan, (iv) tests for age-dependent changes in T cell
subsets and physical activity, and (v) an annual solicitation
for collaborators who wish to suggest new interventions
for evaluation. Mice in the first cohort were exposed
to one of four agents: aspirin, nitroflurbiprofen (NFP),
4-OH-αα
αα
-phenyl-N-tert-butyl nitrone (4-OH-PBN), or nordi-
hydroguiaretic acid (NDGA). An interim analysis was
conducted using survival data available on the date at
which at least 50% of the male control mice had died at
each test site. Survival of control males was significantly
higher, at the interim time-point, at UM than at UT or TJL;
all three sites had similar survival of control females.
Males in the NDGA group had significantly improved
survival (
P
= 0.0004), with significant effects noted at TJL
(
P
< 0.01) and UT (
P
< 0.04). None of the other agents altered
survival, although there was a suggestion (
P
= 0.07) of a
beneficial effect of aspirin in males. More data will be
needed to determine if any of these compounds can extend
maximal lifespan, but the current data show that NDGA
reduces early life mortality risks in genetically heterogeneous
mice at multiple test sites.
Key words: aging; aspirin; longevity; mice; nordihydro-
guiaretic acid.
Introduction
An agent, taken in food or water, that could delay aging would
potentially have a major effect on human health, an effect far
in excess of preventive measures that affect only individual forms
of late-life disease such as cancer or heart disease (Olshansky
et al
., 1990, 2006; Miller, 2002). The published literature contains
sporadic reports (Schneider & Miller, 1998; Schneider & Reed, Jr,
1985) of dietary additives purported to have beneficial effects
in rodent models, but none that has been replicated and accepted
by the scientific community as a reliable and reproducible result.
Stimulated by a consensus conference in 1999 (Warner
et al
.,
2000), the National Institute on Aging (NIA) has developed a
program, the NIA Interventions Testing Program (ITP), to evaluate
agents that are considered plausible candidates for delaying
aging or preventing multiple forms of late-life disease in laboratory
mice. This article presents the central features of the program,
now in its fourth year of operation, along with an interim analysis
of survival data for mice exposed to four agents: aspirin,
nitroflurbiprofen (NFP), 4-OH-
α
-phenyl-N-tert-butyl nitrone
(4-OH-PBN), and nordihydroguiaretic acid (NDGA).
We describe here central features of the ITP protocol: selection
of test agents, simultaneous replication at three test sites, use
of genetically heterogeneous mice, use of both pair-fed and
Correspondence
Richard A. Miller, Department of Pathology and Geriatrics Center, University
of Michigan, Ann Arbor VA Medical Center Ann Arbor, MI 48109-0940, USA.
Tel.: 734 936 2122; fax: 734 647 9749; e-mail: millerr@umich.edu
Richard A. Miller, David E. Harrison and Randy Strong contributed equally to
this study.
†Current address: University of Minnesota, College of Biological Sciences,
St. Paul, MN 55108, USA.
Accepted for publication
26 April 2007
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566
untreated controls, consideration of statistical power, inclusion
of age-sensitive traits, plans for necropsy analysis, and plans for
interim analyses of survival.
Selection of test agents
The NIA makes an annual announcement requesting suggestions
of interventions that may be suitable for inclusion in the ITP
protocol. The most recent announcement in the NIH guide can
be found at grants.nih.gov/grants/guide/notice-files/NOT-AG-07-
003.html, and additional information about the selection
process can be found at www.nia.nih.gov/ResearchInformation/
ScientificResources/InterventionsTestingProgram.htm.
Individual scientists, including those affiliated with govern-
mental, academic, or commercial research groups, are asked to
nominate compounds they think would be worth evaluating in
the ITP, to specify the grounds for their recommendation, and
to provide advice on the best dosage regimen for the study.
These suggestions are considered by an ITP access committee,
whose recommendations are then transmitted to the program’s
steering committee for final approval. The basis for nomination
of a test agent may include a mixture of theoretical ideas and
empirical data. Some compounds are tested because they have
shown promising results in small scale studies of rodents, or
comprehensive studies in worms or flies; others are recommended
because they affect biochemical or physiological processes,
like inflammation or oxidation or stress resistance or insulin
sensitivity, that are thought to be involved in regulation of aging.
Practical considerations prompt greater enthusiasm for inter-
ventions that can be given in food or water and are relatively
inexpensive. Interventions that involve more intensive effort,
such as injection or implantation procedures or environmental
manipulations, can be considered if their biological effect is
thought likely to require only transient or periodic exposure
rather than daily intervention.
Parallel studies at three institutions
The design of the program emphasizes the value of simultaneous
replication at multiple test sites. Investigators interested in
antiaging interventions, either as probes of the aging process
or as a step towards developing preventive medicines for human
use, are likely to have greater confidence in a report that includes
consistent data from multiple sites than they would in the typical
report that includes data from a single research site only. For
this reason, each agent accepted for the ITP is evaluated at three
sites: the Jackson Laboratory (TJL), the University of Michigan
at Ann Arbor (UM), and the University of Texas Health Science
Center at San Antonio (UT). The scientific staff at each of these
three sites have attempted to replicate as carefully as possible
the protocol in use at the other two sites, for example, by ordering
bedding from a single supplier, preparing control and experi-
mental diets from the same supplier and shipped from a single
source, co-ordinating environmental variables such as light/dark
cycles and temperature, etc.
Use of genetically heterogeneous mice
ITP uses mice produced by a four-way crossbreeding scheme,
in which the experimental animals are the progeny of CB6F1
females bred to C3D2F1 males. Each animal in the test population
is thus genetically unique, but each shares half of its non-
mitochondrial genome with every other test mouse; from this
perspective, all the animals are full sibs. This specific four-way
cross (UM-HET3) has been used previously for a series of biomarker
and gene mapping analyses (Jackson
et al
., 1999; Miller
et al
.,
1999a; Harper
et al
., 2003; Hanlon
et al
., 2006), providing some
background information against which ITP data sets can be
compared. In addition, a set of single nucleotide polymorphism
genotyping probes should allow relatively inexpensive quanti-
tative trait locus mapping studies of any trait measured in the
ITP mice. The rationale for using genetically heterogeneous mice
instead of one of the commonly used inbred mouse strains is
to avoid missing true positive effects of agents that might fail
to work in a specific inbred genotype, and to reduce the chance
of giving undue emphasis to an agent that might work only in
the specific inbred stock selected (Committee on Animal Models
for Research on Aging, 1981; Miller
et al
., 1999a,b). Male and
female mice are tested in parallel at each institution.
Use of pair-fed and untreated controls
Each year’s work includes animals exposed to one of a series of
interventions (typically 4, 5, or 6 per year), plus a group of untreated
controls, plus a group of mice whose food supply has been reduced
to a level that allows these ‘pair-fed’ mice to track the weight
trajectory of mice with the lowest mean weight of the experimental
groups. The number of untreated control animals is approximately
twice the number of those in any one of the treatment groups,
to provide increased statistical power for tests of treated against
control mice for life table analyses; use of an unbalanced design
of this kind is a cost-effective way to increase statistical power in
that the same oversampled control group is used for comparisons
against each of the experimental groups. The pair-fed group is
intended to provide a basis for comparison for any agent that
leads to diminished body weight. This comparison may, in some
cases, permit rejection of the idea that an agent increases
lifespan merely by reduction of appetite and thus by inducing
a form of caloric restriction, by allowing a comparison of life table
to mice with a similar weight trajectory but which have not been
exposed to the intervention in question. None of the agents
tested in the first year’s group was found to lead to diminished
mean weights, and mice in the pair-fed group were therefore
removed from the protocol and used for other purposes. Thus
none of the figures or tables in this report include any information
about survival of mice originally assigned to the pair-fed group.
Consideration of statistical power
The ITP standard protocol enrolls 36 female and 44 male mice
in each treatment group, at each of the three test sites, an equal
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567
number in the pair-fed populations, and includes 72 females
and 96 males in the untreated control group. Oversampling of
males is intended to compensate for the larger number of early-
life deaths of male mice due to fighting; the protocol dictates
euthanasia for all mice in a cage containing an animal severely
injured by fighting. This protocol was the result of a formal
power analysis, considering a variety of analytical end-points
and alternative designs, conducted by Drs Andrzej Galecki
(University of Michigan) and Scott Pletcher (Baylor College of
Medicine). According to the power calculations, a design of this
size is sufficient to detect a 10% increase (or decrease) in mean
lifespan with 80% power with respect to unmanipulated
controls of the same sex, even if data from one of the three
test sites are unavailable (e.g. because of a catastrophic failure
of temperature or infection control).
Evaluation of age-sensitive traits
The consensus conference from which the ITP program developed
(Warner
et al
., 2000) included much discussion of what pro-
portion of resources to commit to testing age-sensitive traits in
addition to lifespan. Some researchers advocated evaluation of
a wide range of physiological and biochemical end-points in
each test group, in the hopes of detecting beneficial effects of
agents independent of any effect on longevity, while others
thought that doing so would, because of budgetary constraints,
limit the number of agents (and doses) that could be tested in
any one year. The principal goal of the ITP is to find interventions
that slow aging using extension of maximum lifespan as a
surrogate measure for aging rate. For this reason the ITP group
has decided to include only a small number of mid-life end-points
in its initial testing, reserving more detailed characterization of
multiple systems for later studies of interventions that appear
promising in the initial screens. Even the initial populations
(‘Phase I’ tests), however, are evaluated for three age-sensitive
traits. (i) A sample of mice (50 per test group + 100 controls)
is tested at ages 7 and 18 months for spontaneous locomotor
activity, during a 50-h period in a standard mouse cage, using
a protocol that permits independent assessment of ambulation,
stereotypic movements (in place), and day/night distribution of
activity. (ii) A blood sample taken at 18 months is evaluated
for proportions of four age-sensitive T-lymphocyte subtypes.
(iii) Serum taken at 18 months is evaluated for a series of
hormones including insulin, insulin-like growth factor I (IGF-I),
leptin, and thyroxine (T
4
).
Plans for necropsy analysis
Details of pathological lesions are likely to be of use for evaluation
of any agent that extends lifespan in the ITP protocol, and
potentially for agents that unexpectedly lead to shorter lifespan
than that seen in the control groups, but unfortunately it will
not be possible to learn which agents have these effects until
most of the mice have died. For this reason, the ITP protocol
involves fixation of the bodies of mice found dead (or euthanized
when severely moribund), and their preservation for future study
if necessary.
Plans for interim analyses
We plan to conduct our primary lifespan analysis when we have
a sufficient number of deaths to let us test hypotheses about
maximum lifespan. Analytical plans that include frequent
evaluation of the same evolving data set at multiple intervals
have well-known disadvantages, in particular the inflation of
overall Type I error rate. An interim analysis, however, presents
important advantages, in that it may alert the scientific com-
munity to agents that postpone early-life deaths and may deserve
increased scrutiny for possible effects on aging, age-sensitive
traits, and physiological end-points thought to be important in
the aging process. We have therefore elected to conduct an
interim analysis of survival data at a preselected time-point,
specifically using a data set frozen on the day that 50% of the
control mice have died at the site where this criterion is last met.
Separate interim analyses will be conducted for male and for
female mice, using the survival curve of the corresponding
control population in each case. This report, for example, is
based upon the survival data available on December 30, 2006,
the date on which at least 50% of the male control mice were
dead at each of the three test sites.
Test agents for Cohort 1
For the initial cohort, four agents were selected: aspirin, NFP,
NDGA, and 4-OH-PBN.
Chronic inflammation is associated with many chronic diseases
of aging, and may actually contribute to those diseases. Changes
with age in both adaptive and nonadaptive immunity may alter
both structures and functions of organs and tissues, while
chronic diseases of aging may stimulate inflammation in a
deleterious feedback system. Inflammatory cytokines and other
mediators of inflammation can also be strong near-term predictors
of mortality associated with age-related chronic diseases. An
inflammatory component is involved in the pathogenesis of
many age-associated diseases such as atherosclerosis (Weissberg
& Bennett, 1999), Alzheimer’s disease (Akiyama
et al
., 2000),
many forms of cancer (Masferrer
et al
., 2000), and osteoarthritis
(Pincus, 2001). Aging also is characterized by an elevation in
such proinflammatory cytokines as interleukin 6 (IL-6) and tumor
necrosis factor-
α
(TNF-
α
) (Spaulding
et al
., 1997; Ershler & Keller,
2000), and high levels of IL-6 are powerful predictors of morbidity
and mortality in the elderly (Ferrucci
et al
., 1999; Harris
et al
.,
1999). In addition, caloric restriction, the best-characterized
antiaging intervention, consistently reduces inflammatory
responses. Two anti-inflammatory agents were included in the
first set of ITP test compounds: aspirin and NFP.
Aspirin, a nonsteroidal anti-inflammatory drug (NSAID), also has
antithrombotic and antioxidant properties (Weissmann, 1991;
Shi
et al
., 1999; Vane, 2000). Some of its effects represent
inhibition of cyclooxygenase I (COX-I) and cyclooxygenase II
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568
(COX-II), which synthesize prostaglandins through enzymatic
oxidation of arachidonic acid. At therapeutic doses, aspirin
suppresses production of TNF-
α
by an effect on NF-
κ
B signals
(Shackelford
et al
., 1997). Aspirin may also have indirect effects
on oxidative damage to proteins by acetylation of
ε
-amino
residues, thus blocking inter- and intra-protein crosslinks as
well as glycation reactions (Jones & Hothersall, 1993; Weber
et al
., 1995; Shi
et al
., 1999; Caballero
et al
., 2000). Aspirin at
100 mg kg
–1
per day was found to reduce carbonyl formation
and bityrosine formation in the lens crystallins of diabetic rats,
presumably by an antioxidant effect (Jones & Hothersall, 1993);
formation of advanced glycation end-products was similarly
inhibited. Aspirin at concentrations of 333 mg kg
–1
body weight
per day prevented the development of both cirrhosis and
preneoplastic and neoplastic nodules, but without any directly
associated prevention of fatty changes in male F344 rats (Denda
et al
., 1994).
Nitroflurbiprofen is a nitric oxide (NO)-releasing flurbiprofen
derivative (Ongini & Bolla, 2006). Like the commonly used
NSAIDs such as ibuprofen, NFP has about equal inhibitory
potential against COX-I and COX-II. It crosses the blood–brain
barrier, so may benefit chronic inflammation in the central
nervous system (CNS). The NO-releasing moiety of NFP confers
protection against gastrointestinal toxicity, protects the
cardiovascular system, and enhances the anti-inflammatory
effect, therefore making the drug safer than reference NSAIDs
in conditions requiring chronic administration. Although long-
term anti-inflammatory treatments can sometimes damage
kidney and heart tissues, previous studies have shown that
6–12 months of exposure of mice to NFP did not produce
overt signs of toxicity (van Groen & Kadish, 2005; Brunelli
et al
.,
2007). The dose of 30 mg kg
–1
per day used in previous studies
was fully effective in a variety of experimental paradigms involving
activity in both NO and COX pathways; these data provided
a basis for the dose used in the ITP testing program. Early human
studies have also suggested that NFP may have a favorable
safety profile for the gastrointestinal and cardiovascular system
(Ongini & Bolla, 2006).
Nordihydroguaiaretic acid (NDGA; also called masoprocol)
is a naturally occurring dicatechol produced by the creosote
bush
Larea tridentata
. NDGA has both antioxidant and anti-
inflammatory properties, and resembles other polyphenols,
including resveratrol, that can activate sirtuins and have shown
life-extension properties in metazoan models (Wood
et al
.,
2004). NDGA inhibits arachidonic acid 5-lipoxygenase and its
cytokine-stimulated activation of microglia or macrophages, in
part by suppression of proinflammatory gene expression and
prostaglandin E
2
(PGE
2
) production (West
et al
., 2004). NDGA
therefore might inhibit age-related tissue damage arising from
inflammatory products of arachidonic acid metabolism. One
group has reported that low concentrations of NDGA can extend
mean and median adult lifespan of mosquitoes by 50% (Richie,
Jr,
et al
., 1986). Similarly, NDGA reportedly extends lifespan by
10% in
Drosophila
(Miquel
et al
. 1982) and extends clonal
lifespan in
Neurospora crassa
(Munkres & Colvin, 1976). Only
one report exists of NDGA effects on age-related mortality in
a mammal, a study in which Wistar rats received food containing
0.005% NDGA (Buu-Hoi & Ratsimamanga, 1959); after 2 years
of treatment, eight of 12 remained alive in the NDGA group,
compared to two of 12 control animals. In a separate study,
food containing 0.1% NDGA was reported to slow disease
progression and thus increase lifespan in mice with motor neu-
ron disease caused by a mutation in superoxide dismutase 1
(West
et al
., 2004). Nontransgenic mice fed the same NDGA-
supplemented diet were found to tolerate the drug chronically
and demonstrated a tendency for enhanced motor function
at later ages (K. Hensley, personal communication). These
observations, while fragmentary, provided a rationale for
evaluating the potential effect of NDGA on lifespan and health
in the ITP.
Nitrones, with the general formula X – CH = NO – Y, have
been used for over 30 years in analytical chemistry to trap and
stabilize free radicals. One such agent, PBN (
α
-phenyl-N-tert-
butyl nitrone), has been shown to have potent pharmacologic
activities in experimental models of age-related diseases including
stroke (Floyd, 1990; Zhao
et al
., 1994) and cancer (Floyd
et al
.,
2002; Nakae
et al
., 2003). The principal metabolite of PBN is
its 4-hydroxy derivative,
4-OH-PBN
, which was evaluated in the
ITP. In one study (Edamatsu
et al
., 1995), PBN at 30 mg kg
–1
per day was found to extend lifespan of the short-lived stock
SAM-P8 by about 33%. A second study (Saito
et al
., 1998)
documented a beneficial effect of PBN at about 38 mg kg
–1
per
day on longevity in C57BL/6J mice when treatment was initiated
at 24.5 months of age. In a third study (Sack
et al
., 1996), PBN
was administered at 32 mg kg
–1
body weight per day to Sprague-
Dawley rats, starting at age 24 months, and was shown both
to improve memory retention and to increase median lifespan
from 29.4 to 32.2 months.
We report here the results of a planned interim analysis of
survival, conducted on the data set as it stood on December
30, 2006, the date at which at least 50% of the male control
mice had died at each of the three test sites.
Experimental procedures
Mice
Mouse production, maintenance, and estimation of lifespan
UM-HET3 mice were produced at each of the three test sites.
The mothers of the test mice were CByB6F1/J, JAX stock
#100009, whose female parents are BALB/cByJ and whose male
parents are C57BL/6J. The fathers of the test mice were
C3D2F1/J, JAX stock #100004, whose mothers are C3H/HeJ and
whose fathers are DBA/2J. The first litter from each breeding
cage was discarded, so that all of the experimental animals
would be the product of a second or subsequent pregnancy.
This decision was made to avoid the possibility that mice that
were born to primiparous dams might receive inferior nutrition
or maternal care compared to products of subsequent preg-
nancies. Each test site entered approximately equal numbers of
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569
weanlings each month over a 6-month period. Mice were
weaned into same-sex cages (three males or four females per
cage) at the age of 19–21 days. Sires were not separated from
pregnant dams during the period of pregnancy, to allow sub-
sequent impregnation at postpartum estrous. Mice did not
receive ear punches or toe clips or other identifying marks at
weaning. Each site used diets based on the NIH-31 standard.
For breeding cages, UM used Purina 5008, UT used Teklad 7912,
and TJL used Purina 5K52. For weanlings prior to 4 months of
age, UM used Purina 5001, UT used Teklad 7912, and TJL used
Purina 5LG6.
Mice were housed in plastic cages with metal tops, using
corn-cob bedding, specifically 1/4 inch Bed O’Cobs, produced
by The Andersons, PO Box 114, Maumee, Ohio. Mice were given
free access to water, acidified (pH 2.5–2.7) by addition of
hydrochloric acid, using water bottles rather than an automated
watering system. Mice were housed in ventilated cages, and were
transferred to fresh cages every 14 days, except that at UT mice
were transferred to fresh cages every 7 days. Temperature was
maintained within the range of 21–23
°
C.
At the age of 42 days, each cage was assigned to a control
or test group by use of a random number table. Each mouse was
then briefly anesthetized by isoflurane inhalation administered
either by nose cone or by an instrument designed for small
animal anesthesia. Measures were taken of weight, body length,
and tail length, and an electronic ID chip was implanted by sterile
syringe beneath the dorsal skin between the shoulder blades,
after which the wound was closed by a drop of superglue
(Loctite gel, purchased locally, or Nexaband S/C, purchased from
Abbott Laboratories, North Chicago, IL, USA). UM and UT used
chips purchased from AVID Microchip ID Systems (Folsom, LA,
USA; catalog AVID3002); TJL used chips purchased from Locus
Technology (Manchester, MD, USA; catalog 1D-100A). A
portion of the distal tail (1 cm) was taken and frozen for later
analysis of DNA polymorphisms, after which the mouse was
permitted to awaken from the anesthesia. The duration of
anesthesia was approximately 1–2 min.
Mice received control diet until the age of 4 months, after
which experimental diets were substituted for mice in the NFP,
4-OH-PBN, and aspirin groups. Mice in the NDGA group started
receiving NDGA at the age of 9 months.
At the age of 7 months, a subset of the mice (100 controls
plus 50 of each test group at each site) were evaluated for activity,
using a test in which each mouse is housed individually in a
standard mouse cage for 50 h, while a computer records
episodes of ambulation or movement in place. This test was
repeated, at the age of 18 months, for each live mouse that
had been previously tested. Three weeks after the activity test,
a blood sample was obtained from each mouse that had been
tested for activity; these samples were used for analyses of T
cell subsets and hormone levels that will be reported elsewhere.
Each colony was evaluated for the presence of pathogens
four times each year. The test procedure involves pooling used
bedding from groups of cages, and exposing sentinel mice to
the spent bedding for a period of 6 weeks. Blood samples from
the sentinel mice are then examined for antibodies to mouse
viruses, and the sentinels themselves tested for pinworm eggs.
At UM, the mice are evaluated quarterly for antibodies to mouse
hepatitis virus (MHV), minute virus of mouse (MVM), mouse
parvovirus (MPV), rotavirus (EDIM), and ectromelia, and pinworms
are sought in a cecal exam. In addition, tests are done semiannually
for Sendai, pneumonia virus of mice (PVM), Theiler’s virus
(TMEV, GD VII), reovirus 3 (REO), Mycoplasma pulmonis (MPUL),
lymphocytic choriomeningitis virus (LCMV), mouse adenovirus
(MAV), and polyoma virus. TJL tests for all of these agents
quarterly, plus K virus, Hantaan virus, lactic dehydrogenase
elevating virus, mouse cytomegalovirus (MCMV), and mouse
thymic virus (MTV). In addition, TJL evaluates serum for cilia-
associated respiratory (CAR) bacillus, evaluates tracheal cultures
for
Streptobacillus moniliformis
, and evaluates fecal or intestinal
cultures for
Citrobacter rodentium
and
Salmonella
spp. The
testing protocol at UT is similar, but also includes tests for several
species of
Helicobacter
. All such surveillance tests were negative
at all three sites throughout the experiment.
Removal of mice from the longevity population
Mice were inspected daily, and husbandry staff made a notation
when they found an animal with severe bite wounds, that is,
wounds that were present on more than 20% of the skin or
that were bleeding or infected. If the staff noted continued
evidence of wounding over the next 2-week period, all mice
in the cage were euthanized. Thirty-six mice were removed from
the study for this reason, including 33 males and three females;
of these 36 mice, three were at TJL (0.5% of 654 TJL mice), 20
(3% of 658) at UM, and 13 (2% of 657) at UT. An additional
26 mice were removed because of an accident, typically
either death during chip implantation or failure of the chip to
function (nine at TJL, eight at UM, nine at UT). An additional
51 mice were removed from the longevity protocol when they
were used for another experimental purpose, such as testing
for blood levels of a test agent (33 at UM and 18 at UT). For
survival analyses, all such mice were treated as alive at the
date of their removal from the protocol, and lost to follow-up
thereafter. These mice were not included in calculations of
median longevity.
Estimation of age at death (lifespan)
Mice were examined at least daily for signs of ill health. Mice
were euthanized for humane reasons if they are so severely
moribund that they are considered, by an experienced technician,
unlikely to survive for more than an additional 48 h. A mouse
was considered severely moribund if it exhibited more than
one of the following clinical signs: (i) inability to eat or to drink;
(ii) severe lethargy, as indicated by reluctance to move when gently
prodded with a forceps; (iii) severe balance or gait disturbance;
(iv) rapid weight loss over a period of 1 week or more; or (v)
an ulcerated or bleeding tumor. The age at which a moribund
mouse was euthanized was taken as the best available estimate
of its natural lifespan. Mice found dead were also noted at each
daily inspection.
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Control and experimental diets
The test substances were incorporated into Purina 5LG2 chow,
an irradiated diet containing 4% fat, based on the NIH-31
standard. Experimental diets were administered to the mice
from 4 months of age (except for NDGA, which was initiated
at 9 months of age). Purina prepared batches of food containing
each of the test substances, as well as control diet batches, at
intervals of approximately 4 months, and shipped each batch
of food at the same time to each of the three test sites. NDGA
was purchased from Cayman Chemicals (Ann Arbor, MI, USA)
and mixed with chow at a concentration of 2500 milligrams of
NDGA per kilogram of food. Nitroflurbiprofen was obtained
from NicOx Research Institute (Milan, Italy) and used at a dose
of 200 milligrams NFP per kilogram of food. Aspirin was obtained
from a local supplier, and used at a dose of 21 mg per kg of
food. 4-OH-PBN was synthesized in the laboratory of Robert
Floyd, and used at a dose of 350 mg per kg of food. On the
assumption that each mouse weighs 30 g and consumes 5 g
food per day, the estimated daily doses of these agents would
be NDGA 417; NFP 33; 4-OH-PBN 53, and aspirin 3.3 mg kg
–1
body weight per day.
Statistical methods
Each mouse originally entered into the study was, at the time
of analysis, considered to be in one of three categories: alive,
dead (from natural causes), or culled. Mice were considered to
be culled at the age at which they were no longer subjected
to the mortality risks typical of unmanipulated mice. In some
cases this was because the mouse was removed because of
fighting; in other cases mice died as the result of an accident
(e.g., death when anesthetized for implantation of a radio-emitting
chip). In further cases mice were considered ‘culled’ on the day
in which they received an experimental treatment (such as blood
sampling or injection with an inflammatory agent) to which the
control mice were not exposed. Kaplan–Meier analysis, and
log-rank comparisons among groups consider ‘culled mice’ to
be lost from follow-up on the day at which they were removed
from the longevity protocol, and consider mice alive on the day
of analysis (December 30, 2006) as lost to follow-up at that
time.
Unless stated otherwise, all significance tests about survival
patterns are based upon the two-tailed log-rank test at
P
< 0.05,
with culled mice included up until their date of removal from
the longevity population.
Other statistical tests are described in the text; all
P
values
were two tailed.
Results
Of the 1100 male mice entered into the study (distributed in
roughly equal numbers among the three test sites), 1029
remained unculled at the interim analysis point; of these 1029
mice, 650 (63%) were dead. Of the 869 females originally
enrolled, 827 remained unculled at the analysis point; of these
827 animals, 462 (56%) were dead. The oldest surviving mice
at the analysis point were 32.5 months of age, and the youngest
were 26 months.
Figure 1 shows the survival curves for control males and
females at each of the three test sites. Survival for females is
higher than for males at TJL and at UT (
P <
0.0001 at each site),
but not at UM (
P =
0.32). There is no significant difference
among the three sites in survival for control females, but control
males at UM were significantly more likely to survive than
control males at the other two sites (
P <
0.0001). There were
no significant differences between UT and TJL in survival curves
for either sex.
Table 1 shows median survival times for male and female
control mice at each site, with culled mice excluded from the
calculation. We do not at present understand the basis for the
relatively high mortality risks at TJL and UT compared to UM,
nor why these risks affect male mice but not females.
Figure 2 shows survival curves for male mice, pooling data
across all three sites and including culled animals, for controls
as compared to mice in each of the four experimental groups.
A log-rank test, comparing among all five groups, showed that
the differences among groups were significant at
P
< 0.005,
Fig. 1 Survival curves for male and female mice, in the control group, at each test site. Data are shown only for ages up to 900 days. For males, log-rank
P < 0.0001 for the hypothesis that all three sites show the same survival distribution.
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571
using a calculation stratified by test site. A secondary analysis
then repeated this comparison separately for the male mice at
each test site, and showed a significant difference among experi-
mental groups for male mice at TJL (P < 0.01) and UT (P < 0.04),
but not at UM.
We then repeated the stratified log-rank test, for male mice,
pooling across sites, to compare the control group with each
of the individual test agents. For NDGA, P = 0.0004 for the
pooled data. The NDGA effect was significant for TJL and UT
males at P = 0.02 and P = 0.005, respectively, but was not
significant for males at UM. Figure 3 shows comparisons of
NDGA to control mice for males at each of the three test sites.
There were no significant effects of 4-OH-PBN or of NFP in
male mice, pooling across sites. For aspirin, the log-rank test
showed a trend toward improved survival (P = 0.07, pooled
data), with site-specific P values for TJL, UM, and UT of P = 0.05,
0.64 and 0.17, respectively. We conducted a secondary analysis
using the Wilcoxon–Breslow test (which gives greater weight to
deaths at early time-points) instead of the log-rank test, and
found P = 0.03 for the pooled data set.
Figure 4 shows a comparison among test groups for female
mice, pooling across test sites. The log-rank test did not show
any evidence for significant differences attributable to treatment
group for the female mice.
It is possible that some dietary interventions might cause
a decline in food palatability, leading to a decline in food
consumption and thus to a form of voluntary caloric restriction.
Although neither food consumption nor adiposity was measured
in this pilot study, we did measure weight in each mouse at
ages 6, 12, 18, and 24 months, and at some sites also at ages
1.4, 3, and 4 months, as an indirect measure of nutritional status.
The data are presented separately for each site, and for both
males and females, in Fig. 5. Considering the control groups,
both male and female mice at UM were significantly lighter in
Table 1 Median survival values for male and female control mice
Site Sex Median ± standard error (n)
TJL F 858 ± 7 (93)
UM F 909 ± 4 (86)
UT F 876 ± 4 (96)
TJL M 781 ± 7 (125)
UM M 876 ± 8 (106)
UT M 739 ± 7 (119)
Fig. 2 Survival curves for male mice, pooled across all three test sites, by each
of the four tested interventions, plus controls. Data shown up to the age of
900 days. Log-rank P < 0.005 for the hypothesis that all five treatment groups
show the same survival distribution.
Fig. 3 Survival curves comparing nordihydroguiaretic acid (NDGA) treatment
to control mice, for males only, for each of the three test sites. Data shown
up to the age of 900 days. Log-rank P values refer to the comparison of NDGA
males to control males.
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572
weight than mice at either of the other two test sites at each
age from 6 months onward, even though each site used the
same batches of food prepared and shipped from the same diet
kitchen. The difference between mice in the NDGA and control
groups at UM, seen at 6 months, cannot be attributed to NDGA
effects, because the mice assigned to the NDGA group were
not actually exposed to this agent prior to 9 months of age. At
12 months of age there was a small, but statistically significant
difference between control and NDGA mice at the UM site
(1.9 g, P < 0.02), but this is smaller than the difference seen at
this site (4.4 g) at 6 months of age, that is, prior to NDGA
administration, and so is unlikely to represent a drug effect per se.
There were no significant effects of NDGA at UT or TJL, in male
or female mice. These data do not support the idea that the
beneficial effects of NDGA on survival are mediated by caloric
restriction.
Discussion
Our results suggest that NGDA diminishes mortality risks for
male UM-HET3 mice over the first half to two-thirds of the
expected lifespan. The primary analysis, focused on the pooled
data set, showed that this effect was highly significant, and
confidence in the result was bolstered by noting that the effect
was statistically significant at two of the test sites, and showed
a similar (though nonsignificant) trend at the third test site. It
is noteworthy, however, that the two sites with the strongest
effects of NDGA were those at which the control male mice
had the highest risk of mortality. The effect of NDGA, at this
interim time-point, also seems to be limited to male mice, contrary
to our working hypothesis that any beneficial effects of test
agents would be seen in mice of both sexes. Based on the body
weight data, it seems very unlikely that the effect of NDGA is
mediated by caloric restriction. None of the other three tested
agents had a significant effect on survival at this interim test
point, although there is a suggestion that aspirin might have
a small benefit for early survival in males. Because of cost
considerations, we were only able to test each compound at a
single dose, and the dose selected was in each case based on
only a limited amount of previous experience with the agent
or its congeners in rodents. It is possible that different results,
potentially including a positive effect on mid-life survival risks,
might have been seen at doses higher or lower than those used
in this study.
Although there have been many prior reports in which agents
added to rodent chow have been seen to extend median or
maximum lifespan (see Schneider & Miller, 1998; Schneider &
Reed, 1985 for reviews), so far as we know none of these have
been replicated, either at the original test site or at a different
site. The design of the ITP features simultaneous replication
at three independent sites, and it is noteworthy that the NDGA
effect indeed does seem to be similar at all three sites, with a
statistically significant effect at the two sites with the largest
number of recorded deaths at this interim time point.
We wish to emphasize that these results do not address the
central goal of the ITP, which is to determine if any of the test
agents slow aging to an extent sufficient to increase maximum
lifespan. There are circumstances in which an intervention, such
Fig. 4 Survival curves for female mice, pooled across all three test sites, by
each of the four tested interventions, plus controls. Data shown up to the
age of 900 days.
Fig. 5 Mean weight as a function of age, comparing nordihydroguiaretic acid
(NDGA) to control mice, at each of the three test sites. Top panel shows males,
and bottom panel shows females. Each symbol shows mean ± SEM for 24
60 mice in each NDGA group, and for 46–125 mice in each control group
(depending on age and site).
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as exercise (Holloszy, 1993), has been shown to increase median
lifespan or some other index of early-life mortality risks, without
any effect on maximum lifespan or an equivalent index of survival
among the oldest test subjects. NDGA might, for example, act
by postponing one or more specific forms of lethal early-life
illness. We anticipate that 90% of the control population is likely
to have died by about 32 months of age, and that the youngest
group of mice in our study will have reached this age by July
2007. At this point, it will be possible to assess the effect of
treatments on the proportion of mice that live past the age at
which 90% of the population has died; a significant effect using
a quantile test (Wang et al., 2004) might then support the idea
that one or more of the agents may retard aging. No such
conclusion would, however, be warranted at this stage.
We do not understand why median survival for male mice
is higher at UM than at UT or TJL, nor why the factors that
contribute to this difference among sites affect males but not
females. Historical control data are available from three prior
longevity studies performed on male and female virgin UM-
HET3 mice at the University of Michigan, from which median
survival values can be calculated. One such study (Jackson et al.,
2002) found median survival of 787 days for males and 818 days
for females, excluding the male mice that died at early ages from
urinary syndrome. A second study (Miller & Chrisp, 1999) had
median survival of 829 days for males and 836 days for females.
The third, and largest, population (Harper et al., 2004) had
median levels of 862 days for males and 864 days for females.
Comparison of these historical values to the data in the present
study is not straightforward, because of differences in protocols,
including, for example, the elimination of first litters from the ITP
study. The median value for ITP control males at UM (876 days)
is similar to that seen in the most recent of the prior populations,
and the median levels for control males at UT and TJL (739 and
781 days) are somewhat lower than those seen in any of the
three UM-HET3 lifespan experiments, all of which were con-
ducted at UM. In contrast, the values for female control mice
(858, 876, 909 for the three ITP sites) are similar to or slightly
higher than those seen in the previous UM-HET3 populations,
suggesting that the variation among the male populations is
unlikely to represent any systematic problems with husbandry
operations or standards of health care in any of the colonies.
Each of the three test sites used a different variation of the NIH-
31 diet for breeding cages, and for weanling mice prior to the
initial exposure to experimental and control diets, and it is possible
that variations in vitamin levels, salts, and protein sources at
these early ages might have contributed to differences among
sites in male survival and male and female weight trajectories.
There are a small number of other known inconsistencies among
the sites, such as the use of different brands of implanted
microchips, and the schedule of cage changes (14 days at TJL
and UM, 7 days at UT), and there are likely to be changes in
other environmental variables (noise levels, light or temperature
gradients, odors) that are not apparent to investigators but
could potentially affect health of the mice. The dramatic
variation among sites in body weights of both male and female
animals, despite the use of rodent chow shipped to each site,
in parallel, from a single source, testifies to the presence of
uncontrolled environmental factors with potent effects on key
metabolic endpoints, and thus to the importance of replication
of life table data at independent test facilities.
NDGA is a product of the creosote bush, L. tridentata,
which grows in the southwestern USA and northern Mexico. It
has a wide variety of uses in folk medicine including treatment
of infertility, rheumatism, arthritis, diabetes, gallbladder and
kidney stones, pain, and inflammation. It is a potent antioxidant
and was widely used in the 1950s as a food additive to prevent
spoilage, until it was banned because of reports of toxicity in
the 1960s (Arteaga et al., 2005). It has been reported to increase
lifespan in mosquitoes, fruit flies, and rats and to prolong the
survival of the G93A mouse model of amyotrophic lateral sclerosis
(Buu-Hoi & Ratsimamang, 1959; Miquel et al., 1982; Richie et al.,
1986; West et al., 2004). In addition to its antioxidant prop-
erties, there are several actions of NDGA that could lead to
beneficial effects. NDGA has a spectrum of activities similar to
that of other naturally occurring biphenolic compounds such as
resveratrol. It is widely reported to be a potent anti-inflammatory
agent through its inhibitory action on 5-lipoxygenase to
prevent leukotriene synthesis (e.g. West et al., 2004). It has been
reported to enhance glucose clearance and insulin sensitivity
and to dramatically reduce serum triglycerides in a rat model
of type II diabetes (Reed et al., 1999). The latter effect may be
related in part to NDGA’s reported ability to block fatty acid
synthesis in adipocytes through inhibition of fatty acid synthase
(FAS) and to inhibit lipoprotein lipase (Park & Pariza, 2001; Li
et al., 2005). In numerous studies, NDGA has been reported to
have anticancer activity through its action as a lipoxygenase
inhibitor (e.g. McDonald et al., 2001; Hoferova et al., 2004;
Nony et al., 2005). It has also been reported to suppress growth
of breast cancer cells through inhibition of the function of two
receptor tyrosine kinases (RTK), the insulin-like growth factor
receptor (IGF-R) and c-erbB2/HER2/neu (HER2/neu) (Youngren
et al., 2005). NDGA may act to prevent deleterious effects of
aging on the brain. Expression of 5-lipoxygenase is elevated with
age and has been proposed to play a role in the pathobiology
of aging-associated neurodegenerative diseases (Manev et al.,
2000; Qu et al., 2000). Indeed, NDGA has been reported to be
effective in preventing neuronal death and cognitive deficits
resulting from forebrain ischemia-reperfusion injury (Shishido
et al., 2001).
Although we do not yet know whether NDGA will extend
maximum lifespan, our data show that this compound does
diminish mortality risks among genetically heterogeneous, male,
adult mice prior to the last third of the expected lifespan, and
thus suggest that further studies of its effects on development
and aging are likely to be worthwhile. Such studies might
usefully include evaluations of effects of NDGA on multiple
markers of health in middle-aged mice, including analyses of
CNS function, T cell subsets, collagen cross-linking, lens turbidity,
and early signs of neoplastic and degenerative illnesses. Evaluations
of possible modes of action of NDGA, including measurements
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574
of hormonal, oxidative, and metabolic indices, as well as studies
of patterns of gene expression and protein profiles, and the
function of key tissues and organ systems, could help to test
specific hypotheses about NDGA-sensitive pathways relevant to
the aging process and pathogenesis of late life illnesses. Studies
of other compounds with structures or pharmacologic profiles
similar to NDGA could also be informative. Our data also provide
a rationale for evaluation of NDGA effects on mortality risks in
other varieties of mice, including other genetically heterogeneous
stocks, and potentially for parallel studies using short-lived
primates. Lastly, administration of NDGA to mice whose aging
rate is unusually slow, for example mice on calorically restricted
or methionine-restricted diets, or pituitary dwarf mutants, could
help to determine whether the pathways by which NDGA
affects survival overlap those involved in other genetic or non-
genetic models of lifespan extension in mice.
Acknowledgments
This work was supported by NIA grants AG022303 (RAM),
AG025707 and AG022308 (DEH), and AG022307 and AG13319
(RS). We wish to thank Vivian Diaz, Elizabeth Fernandez, Melissa
Han, Patricia Harrison, Bill Kohler, Pam Krason, Jessica Sewald,
and Maggie Vergara for technical support. We thank Scott
Pletcher and Andrzej Galecki for assistance in power analysis
and study design.
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    • "Young (3 month-old), middle-aged (12 month-old), and old (20 month-old) male mice hearts were used and divided into six experimental groups: (1) Ischemia/reperfusion young (I/R-Y, n = 8); (2) Ischemic postconditioning young (PostC-Y, n = 7); (3) Ischemia/reperfusion middleaged (I/R-MA, n = 8); (4) Ischemic postconditioning middle-aged (PostC-MA, n = 9); (5) Ischemia/reperfusion old (I/R-O, n = 8); and (6) Ischemic postconditioning old (PostC-O, n = 7). For the middle-aged group, mice should be at least 10 month-old; the upper age limit for the middle-aged group is typically 14–15 month-old [20, 21]. For this reason we decided to use 12 month-old as middle-aged mice, and 20 month-old as old mice. "
    [Show abstract] [Hide abstract] ABSTRACT: Thioredoxin-1 (Trx-1) is part of an antioxidant system that maintains the cell redox homeostasis but their role on ischemic postconditioning (PostC) is unknown. The aim of this work was to determine whether Trx-1 participates in the cardioprotective mechanism of PostC in young, middle-aged, and old mice. Male FVB young (Y: 3 month-old), middle-aged (MA: 12 month-old), and old (O: 20 month-old) mice were used. Langendorff-perfused hearts were subjected to 30 min of ischemia and 120 min of reperfusion (I/R group). After ischemia, we performed 6 cycles of R/I (10 s each) followed by 120 min of reperfusion (PostC group). We measured the infarct size (triphenyltetrazolium); Trx-1, total and phosphorylated Akt, and GSK3β expression (Western blot); and the GSH/GSSG ratio (HPLC). PostC reduced the infarct size in young mice (I/R-Y: 52.3 ± 2.4 vs. PostC-Y: 40.0 ± 1.9, p < 0.05), but this protection was abolished in the middle-aged and old mice groups. Trx-1 expression decreased after I/R, and the PostC prevented the protein degradation in young animals (I/R-Y: 1.05 ± 0.1 vs. PostC-Y: 0.52 ± .0.07, p < 0.05). These changes were accompanied by an improvement in the GSH/GSSG ratio (I/R-Y: 1.25 ± 0.30 vs. PostC-Y: 7.10 ± 2.10, p < 0.05). However, no changes were observed in the middle-aged and old groups. Cytosolic Akt and GSK3β phosphorylation increased in the PostC compared with the I/R group only in young animals. Our results suggest that PostC prevents Trx-1 degradation, decreasing oxidative stress and allowing the activation of Akt and GSK3β to exert its cardioprotective effect. This protection mechanism is not activated in middle-aged and old animals.
    Full-text · Article · Mar 2016 · Molecular and Cellular Biochemistry
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    • "Some of them converge on exercise and diet and their associated pathways. In particular , one of the recurring themes is that of pathways related to energy and nutrient sensing and production [32] , and dietary restriction has emerged as the most robust means of extending life spans and health spans alike [26] . Dietary restriction may be the best path towards this goal, even though its long-term effects on humans are ultimately unknown. "
    [Show abstract] [Hide abstract] ABSTRACT: Research into ageing and its underlying molecular basis enables us to develop and implement targeted interventions to ameliorate or cure its consequences. However, the efficacy of interventions often differs widely between individuals, suggesting that populations should be stratified or even individualized. Large-scale cohort studies in humans, similar systematic studies in model organisms as well as detailed investigations into the biology of ageing can provide individual validated biomarkers and mechanisms, leading to recommendations for targeted interventions. Human cohort studies are already ongoing, and they can be supplemented by in silico simulations. Systematic studies in animal models are made possible by the use of inbred strains or genetic reference populations of mice. Combining the two, a comprehensive picture of the various determinants of ageing and 'health span' can be studied in detail, and an appreciation of the relevance of results from model organisms to humans is emerging. The interactions between genotype and environment, particularly the psychosocial environment, are poorly studied in both humans and model organisms, presenting serious challenges to any approach to a personalized medicine of ageing. To increase the success of preventive interventions, we argue that there is a pressing need for an individualized evaluation of interventions such as physical exercise, nutrition, nutraceuticals and calorie restriction mimetics as well as psychosocial and environmental factors, separately and in combination. The expected extension of the health span enables us to refocus health care spending on individual prevention, starting in late adulthood, and on the brief period of morbidity at very old age.
    Full-text · Article · Dec 2015 · Gerontology
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    • "Optimally, the functional role and measurement technique for these molecular processes will be translatable into human studies. This rate-based BM approach may be particularly promising in combination with what is currently the most robust program for testing proposed interventions for extension of maxLS in mammals: the National Institute on Aging's (NIA) Interventions Testing Program (ITP; Miller et al. 2007). These studies are the current gold standard for evaluating changes in lifespan (LS) in genetically heterogeneous mice but are timeand resource-intensive, limiting the number of interventions that can be "
    [Show abstract] [Hide abstract] ABSTRACT: Combating the social and economic consequences of a growing elderly population will require the identification of interventions that slow the development of age-related diseases. Preserved cellular homeostasis and delayed aging have been previously linked to reduced cell proliferation and protein synthesis rates. To determine whether changes in these processes may contribute to or predict delayed aging in mammals, we measured cell proliferation rates and the synthesis and replacement rates (RRs) of over a hundred hepatic proteins in vivo in three different mouse models of extended maximum lifespan (maxLS): Snell Dwarf, calorie-restricted (CR), and rapamycin (Rapa)-treated mice. Cell proliferation rates were not consistently reduced across the models. In contrast, reduced hepatic protein RRs (longer half-lives) were observed in all three models compared to controls. Intriguingly, the degree of mean hepatic protein RR reduction was significantly correlated with the degree of maxLS extension across the models and across different Rapa doses. Absolute rates of hepatic protein synthesis were reduced in Snell Dwarf and CR, but not Rapa-treated mice. Hepatic chaperone levels were unchanged or reduced and glutathione S-transferase synthesis was preserved or increased in all three models, suggesting a reduced demand for protein renewal, possibly due to reduced levels of unfolded or damaged proteins. These data demonstrate that maxLS extension in mammals is associated with improved hepatic proteome homeostasis, as reflected by a reduced demand for protein renewal, and that reduced hepatic protein RRs hold promise as an early biomarker and potential target for interventions that delay aging in mammals.
    Full-text · Article · Nov 2015 · Aging cell
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