Aging

By Mark R. Bartlett, PhD

Agenomic understanding of aging
is paving the way to identify
interventions that can have significant
impact on the aging process. The
polymorphic nature of aging indicates
that any anti-aging strategy has to start
with a better understanding of genes
that affect tissue viability.
Our anti-aging approach has always
centered on the foundation of good
macro and micro-nutrition, including
the consumption of plentiful plantbased
antioxidants and phytonutrients.
However recent advances, especially
with the mapping of the human genome
and the subsequent development
of DNA microarrays provide (a) an
opportunity to explore the mechanisms
of aging and (b) the tools to begin addressing
aging at its most fundamental
level. We believe that if we are to
widen the gap between chronological
and biological age we must better
understand the role of gene expression
in aging and how dietary ingredients
interact with gene expression in a positive
way
Introduction
Aging is not an episodic process;
rather, it is the consequence of a continuum
of cumulative damage occurring
at the molecular, cellular and tissue
levels. The rate of aging rests on factors,
internal and external, that can either
positively or negatively influence the
balance between tissue preservation or
repair, and damage. Attenuation of aging
is entirely dependent on mitigating
such molecular damage by augmenting
protection and compensatory repair
mechanisms or slowing the degenerative
processes. In a practical sense we’ve all
probably witnessed clear discrepancies
between chronological and biological
age in certain individuals. And while it
has been proposed that genetic factors
contribute to the phenomenon of
people looking old, or young, for their
years, most of us intuitively suspect that
there are some environmental components
over which we wield a certain
amount of control. Therefore, if we are
to widen the gap between chronological
and biological age, we must understand
the various mechanisms involved in
aging and devise effective strategies that
turn these mechanisms in favor of tissue
protection or repair and regeneration.
The question we are asking is: what
are the lifestyle factors and nutritional
components that may assist us in taking
control of our own aging process so that
we can age healthily and reduce agerelated
morbidity?
Macro and Micro Nutrition
A major factor in healthy aging
involves what, and how much, we eat.
There is ample evidence that poor
nutrition, which includes overeating and
poor nutrient density, is linked with an
increased risk for many degenerative
diseases including heart disease, diabetes
and cancer. It is now also becoming
clear that even marginal micronutrient
deficiencies over time lead to accelerated
aging.1
Deficiencies in several
different micronutrients including folic
acid, vitamin D and Magnesium lead
to DNA damage and accelerate agerelated
mitochondrial dysfunction;
which in turn leads to further oxidative
damage to DNA, RNA, proteins and
membrane lipids leading to functional
decline in mitochondria, cells, tissues
and organs. Since multiple studies and
extensive government-commissioned
surveys point to the widespread nature
of inadequate dietary intakes of fruits
and vegetables (and therefore vitamins
and minerals)2 it seems prudent that all
individuals either improve their diets or
supplement their diets with a multivitamin
mineral supplement to ensure
that there are no shortfalls in essential
nutrients.
Free-radical Biology and Antioxidants
A leading hypothesis of aging is based
on the free radical theory of aging by
Harman3 who argued that oxygen-free
radicals produced during normal cellular
respiration cause cumulative damage
to molecules which progressively leads
to loss of functionality of the organism.
Since Harman’s theories were first
proposed, a huge body of literature has
emerged providing evidence that free
radicals and oxidative stress are involved
in many disease states, especially agerelated
degenerative disease. Although
oxidative stress may be a significant
factor associated with aging, it is clearly
not the only contributor and recently
evidence is emerging to support the
concept that vitamins, minerals, and
mediating the survival of an organism.
Since the development of DNA microarrays
that allow scientists to measure
the work output of all of the genes in a
single experiment, it is now possible to
rapidly explore the differences in the
expression of multiple genes between
two or more biological conditions in a
single experiment. Our research andphytonutrients not only fight free radicals,
but they exert perhaps even more
powerful anti-aging effects through a
non-antioxidant role. Phytonutrients,
many of which are antioxidants, also
influence the expression or activity of
factors involved in aging including, for
example, sirtuins, AMPK, NFKB and
PGC-1 alpha to name a few.4-6 Thus it
is becoming increasingly clear that the
phytonutrients we thought were merely
antioxidants are also capable of modulating
gene expression.
Gene Expression Science
It is clear that a nutraceutical approach
to anti-aging must take into
account the polymorphic nature of
aging, and that the crosstalk among
multiple genes plays a more important
role than the action of a single gene in

development team at Nu Skin became
intrigued with the possibility of measuring
the aging process objectively at the
genetic expression level after reading
some of the exceptional work published
by Weindruch, Prolla and colleagues
(LifeGen Technologies, LLC) (LGT)
wherein a powerful technique of differential
expression analysis was being
used to conduct genome-wide searches
for consistent changes in gene expression
patterns that occur during the aging
process.7, 8
Studies using whole-genome transcriptional
profiling typically identify
thousands of genes that are changed
in expression with age. Since many
of these age-related changes are not
universal, but rather are specific to the
genetic background of the organism
being studied, LGT identified biomarkers
of age across seven strains of mice
(5 months vs. 28-30 months old) so
that only the most conserved relevant
patterns of age-related gene expression
markers were considered. Moreover,
these analyses were performed in
three tissues (heart, cerebral cortex and
gastrocnemius) and real-time quantitative
PCR was used to confirm a panel
of 10-20 genes in each tissue. Data
generated from such a model are not
only of a higher standard of rigor, but
they are more likely to be applicable to
human aging as well. Using this approach
statistically robust patterns, or
signatures, of youthful and older gene
expression have emerged which enabled
us to essentially measure aging at
the genetic level. The possibility now
existed to screen for ingredients or formulations
for their ability to retard the
aging process. This is the procedure that
Pharmanex has adopted in collaboration
with LGT to target aging at the source,
gene expression, in an approach that we
call ageLOC science.
Microarrays, Databases and
Bioinformatics as a Guide to Product
Development
In our first screening experiment
certain ingredients emerged for their
abilities to reset gene expression to that
of a more youthful pattern. A particular
preparation of pomegranate, for example,
was the most effective compound tested,
opposing 32-65% of the overall aging
change depending on the tissue studied.
Other ingredients and formulations also
emerged as having potent effects on gene
expression that attenuated age-associated
patterns of expression. The results of
our first round of screening provided an
important insight into ingredients that influence
gene expression in a positive way
and served as an important foundation to
further product development.
In addition to helping identify individual
gene expression signatures associated
with aging, DNA microarray technology
in conjunction with gene databases
and bioinformatics can also be used to
identify the expression levels of groups
of genes that work together to serve a
particular metabolic pathway. We added
the use of such pathway analyses to our
repertoire of microarray-related gene
tools to help further guide our product
development in formulating anti-aging
products by applying it to the concept
of age-related vitality loss. One of the
earliest manifestations of human aging
is a decline in vitality. Mitochondrial
dysfunction associated with aging yields
bioenergetic defects within the cell9
that
exert profound effects on physical and
mental vitality. Our goal was to identify
and target functional gene clusters associated
with mitochondrial aging.
In our attempt to identify these gene
pathways we found that of 20,687 gene
transcripts measured by the Affymetrix
Mouse Genome array, 1241 were associated
with the mitochondria by pathway
ontology (using a gene ontology database).
After our murine feeding studies
and microarray screening we found that
172 of these genes changed in expression
during aging in cerebral cortex
tissue. In gastrocnemius tissue 220 genes
changed which age. Cs-4 opposed the
age-related changes in 52 of these genes
(P<0.05). In addition, Cs-4 opposed the
effects of aging in several gene ……
bioenergetic defects within the cell9
that
exert profound effects on physical and
mental vitality. Our goal was to identify
and target functional gene clusters associated
with mitochondrial aging.
In our attempt to identify these gene
pathways we found that of 20,687 gene
transcripts measured by the Affymetrix
Mouse Genome array, 1241 were associated
with the mitochondria by pathway
ontology (using a gene ontology database).
After our murine feeding studies
and microarray screening we found that
172 of these genes changed in expression
during aging in cerebral cortex
tissue. In gastrocnemius tissue 220 genes
changed which age. Cs-4 opposed the
age-related changes in 52 of these genes
(P<0.05). In addition, Cs-4 opposed the
effects of aging in several gene ontology
pathways. In essence we were able to
identify mitochondrial-related nuclear
encoded genes which changed consistently
in expression with age, or mitochondrial
youth gene clusters (YGC).
A number of natural compounds were
screened for their ability to reset the expression
profile of these genes to a more
youthful level. One ingredient, Cordyceps
sinensis Cs-4 (Cs-4)10 was shown
to markedly attenuate these age-related
gene expression changes in the mitochondria,
suggesting its potential use as
a therapeutic intervention of age related
vitality loss. Ongoing studies are utilizing
this technique to investigate the
effects of a variety of natural ingredients
in brain, muscle and other tissues, but
the sum of such explorations so far, into
the ability of certain natural products to
influence gene expression in a positive
way, has provided strong guidance to our
product development process targeted
at attenuation of the aging process.
Functional Studies
The techniques that we have used
for studying gene expression have not
disappointed us in their promise as tools
explore the mechanisms of aging and
drive us towards meaningful product
development strategies. We see great
promise in the ability of certain nutraceutical
ingredients and formulations to
have a marked effect on gene expression
to oppose age-related changes. The
next logical step is to support these gene
expression data with functional studies.
Indeed, the ingredients that we selected
for gene expression screening were
based on promising functional studies
that had already been performed. However,
to close the circle, we have followed
up these promising gene expression
data with further functional, safety
and efficacy studies in both animals
and humans. Some of these studies are
already completed and have provided
positive correlation and confirmation of
the gene expression data; other studies
are still underway.
Conclusion
Since aging can be considered as a
function of how genes respond to diet
and environmental perturbations through
gene expression while maintaining their
primary function to survive, we chose to
exploit a gene expression approach to
screen several nutraceutical ingredients
and formulations for their effects on
retarding the aging process. We called
this approach ageLOC science. Our
first foray into this approach involved
targeting age-related vitality loss through
an exploration of the gene expression
changes involved in mitochondrial aging.
We identified tissue-specific functional
YGCs, or signatures of gene expression
changes associated with mitochondrial
aging and screened for ingredients that
restored the more youthful pattern of
gene expression. Functional studies have
confirmed the promise offered by the
gene expression study results. It is our
opinion that while a foundation sound of
nutrition and a positive lifestyle are key
to healthy aging and compression of morbidity,
there is much to be gleaned from
an understanding of gene expression as it
relates to the aging process as we pursue
the goal dying young – as late in life as
possible.

Dr. Bartlett has degrees in Biochemistry and
Organic Chemistry from the Australian National University
and a Ph.D. in Immunology and Cell Biology
from the John Curtin School of Medical Research in
Canberra, Australia. In Australia he conducted research
on cardiovascular disease with an emphasis
on the role of reactive oxygen species and free
radicals. He also studied the role of blood platelets
in heart disease, and helped publish the first scientific
report of a biochemical link between cigarette
smoking and atherosclerosis.
Later Dr. Bartlett became interested in autoimmune
inflammatory diseases and examined a
number of plant-derived substances for their ability
to inhibit graft rejection, inhibit cancer metastasis
– or spreading – as well as natural products that
were able to inhibit autoimmune disease.
Before joining Pharmanex Dr. Bartlett was a visiting
scientist at the National Institutes of Health, National
Cancer Institute in Bethesda, MD where, at the
National Cancer Institute, he investigated the interaction
of T-cells with the blood vessel wall, and the
role of various adhesion molecules that are used by
these cells to communicate with one another. He
is currently the Vice President of Global Research
and Development for Pharmanex.