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We
continue our discussion series (initiated
last year) with PGC-1, Calorie
Restriction and Aging. As
always,
we welcome your written comments
- these will be printed in subsequent
issues of our Newsletter. Our many thanks to Dr. Norm Wolf
for editing this newsletter segment.
PGC-1,
Calorie Restriction and Aging
Holly M.
Brown-Borg University of North Dakota
School of Medicine and Health Sciences
and J. Christopher Corton
National Health and Environmental
Effects Research Lab
US Environmental Protection Agency
Transcription regulating factors have
received a fair amount of attention in
the last few years as we continue to
search for mediators of the aging
process. The peroxisome proliferator-activated
receptor g coactivator 1 (PGC-1) is a
family of transcriptional coactivator
proteins involved in nutrient transport
and metabolism, xenobiotic metabolism
and stress resistance. The roles of
PGC-1 in aging and longevity are just
beginning to emerge as the nuclear
hormone receptor (NR) field develops and
our understanding of molecular
regulatory mechanisms grows. Obvious
connections of PGC-1 to current research
interests in aging include calorie
restriction, oxidative stress, stress
resistance, energy metabolism,
insulin-like growth factor-1
(IGF-1)/insulin signaling and longevity
to name a few. For the purposes of this
discussion however, the focus will be
the role of PGC-1, affected nuclear
receptors (PPAR, RXR, CAR) and calorie
restriction (CR) in aging.
Like other
coactivators, proteins in the PGC-1
family modulate transcription by
bridging interactions between DNA
binding transcription factors and the
transcriptional machinery in the absence
of direct interaction themselves.
Activation of nuclear receptors by PGC-1
can be ligand-dependent or –independent,
both of which result in increased
transcription initiation of target
genes. Members of the PGC-1 family,
PGC-1a and PGC-1b, share homology within
certain regions of their protein
sequences that relates to their
coactivator function. Differences in
structure and sequence affect their
interactions with nuclear receptors.
Many of these structural domains are
evolutionarily conserved suggesting that
PGC-1 plays an important role in overall
transcriptional regulation.
PGC-1
regulates several nuclear receptors that
are critical modulators of physiological
processes. There are four classes of
NR, although most of those regulated by
PGC-1 and known to be involved in
responses to nutrient deprivation belong
to class II (including constitutive
activated receptor (CAR), farnesoid X
receptor (FXR), liver X receptor (LXR),
peroxisome proliferators-activated
receptor (PPAR), pregnane X receptor (PXR),
retinoid X receptor (RXR), thyroid
hormone receptor, vitamin D receptor)
family members that heterodimerize to
RXR. PGC-1 also interacts with ERRa,
glucocorticoid receptor, HNF-4a, and the
estrogen receptor.
Several
investigators have suggested that PGC-1
may be a master regulator of
mitochondrial biogenesis and function
thus, playing a central role in energy
homeostasis. Many of the NR regulated
by PGC-1a are altered following CR and
affect expression of genes involved in
energy utilization and stress
responses. PGC-1a is regulated by
hormones that respond to alterations in
glucose concentrations and environmental
stimuli. For example, insulin negatively
regulates gluconeogenesis and fatty acid
b-oxidation at least in part via
suppression of PGC-1a promoter
activity. PGC-1a and PGC-1b gene
expression is induced by fasting and CR,
coordinately regulating genes involved
in gluconeogenesis and fatty acid
b-oxidation. PGC-1a promoter activity
is also activated by glucagons, thus
increasing PGC-1a expression. In
addition, many peroxisome proliferator-inducible
genes are regulated by GH in part via
Stat5b and PPARa interactions.
Several
other factors affect PGC-1 expression
and activity in tissues. Calorie
restriction and fasting lead to low
insulin and increased PGC-1 a expression
in liver. Exercise also increases
PGC-1a expression via activation of
calcium/calmodulin-dependent protein
kinase IV and calcineurin and via MAPK
pathways. Inactivation of insulin
receptor increases PGC-1a expression
while chronic activation of Akt in mouse
heart decreases PGC-1a expression.
Conditions that induce mitochondrial
biogenesis such as nitric oxide increase
PGC-1a promoter activity. PGC-1a
activity may also be regulated post-transcriptionally
by three types of modifications: 1) p38
MAPK phosphorylation which increases
activation of fatty acid b-oxidation
genes; 2) arginine methylation by
protein arginine methyltransferase
(another NR coactivator) which
facilitates activation of genes involved
in mitochondrial biogenesis; and 3)
acetylation which alters the expression
of gluconeogenic and glycolytic genes.
Much of
what is known about PGC-1, nuclear
receptors and CR involves PPARa. PPARa
is activated by peroxisome proliferators
including hypolipidemic agents and
several compounds found endogenously
(e.g., fatty acids or dietary components
(pristanic acid, phytanic acid,
resveratol, oleylethanolamine)). PPARa
is involved in carbon source
utilization. PPARa is upregulated in
several tissues by fasting and CR and
downregulated in pancreatic b-cells by
glucose. PPARa-null mice exhibit
defects in the ability to regulate genes
involved in fatty acid b-oxidation and
ketogenesis in parallel with difficulty
maintaining proper levels of blood
glucose and ketone bodies. PPARa
activation has been shown to regulate a
subset of CR-responsive genes in the
liver involved in fatty acid metabolism,
inflammation and cell growth. In
addition, like CR, PPARa regulates
responses to diverse forms of stress.
Exposure to PPARa-agonists decreases
cellular damage, increases tissue repair
and decreases mortality following both
physical and chemical stressors.
Another
nuclear receptor regulated by PGC-1 that
is likely involved in the beneficial
effects of CR is CAR. CAR regulates
phase I, phase II and phase III pathways
of oxidative metabolism, conjugation and
transport of xenobiotics. This receptor
exhibits constitutive activity but can
also be further activated by exogenous
compounds. CAR controls genes involved
in thyroid hormone metabolism induced by
fasting and there is evidence that CAR
controls xenobiotic metabolism genes
that regulate thyroid hormone levels
during CR. Although this discussion is
limited to mammalian aging, it is of
particular signficance that long-living
C. elegans daf-2 mutants exhibit
enhanced xenobiotic metabolism similar
to rodents subjected to CR suggesting a
common pathway involved in stress
resistance and longevity.
The
molecular mechanisms that underlie the
beneficial effects of CR are not well
understood. However, several
laboratories have focused efforts on the
insulin/IGF-1 pathway as the
physiological effects of CR in rodents
appear to overlap with the effects of
reduced signaling through this pathway.
Calorie restriction decreases plasma
concentrations of growth hormone, IGF-1,
insulin and glucose, thus reducing
signaling, enhancing insulin sensitivity
and extending life span. Reductions in
GH, IGF-1and/or insulin signaling via
engineered or spontaneous mutations
significantly extend life span in
multiple species. Some of the
expression overlap between dwarf and CR
phenotypes in liver is found in genes
that are regulated by PGC-1. Comparison
of survival plot slopes between long
living mice with underlying defects in
GH/IGF-1 signaling and those subjected
to CR reveal differently shaped curves
suggesting more of a delayed aging in
mutant mice versus a decelerated aging
in CR mice. In addition, genes
regulating fatty acid b-oxidation and
fat metabolism are closely linked to
insulin action. Therefore, the
PGC-1-regulated genes, specifically
through PPAR and RXR, are likely at the
heart of this issue.
Several
papers published in the past year shed
some light on the GH-IGF-1-insulin
relationship with calorie restriction,
the role of the PPARs and aging. First,
the Corton laboratory demonstrated that
some of the long-living dwarf mice
constitutively express several PPARa-regulated
genes. To understand the phenotypic
similarities between dwarf mice (Snell)
and peroxisome-proliferator (PP)-treated
wild type mice, the transcriptional
profiles under these two conditions were
compared and revealed a significant
overlap (40%) in gene expression. Genes
with known roles in fatty acid
metabolism that were induced by PP
treatment of wild type mice were found
to be constitutively upregulated in
dwarf mice including Acox1, Cyp4a10,
Cyp4a14, Dci, Fabp4, Ech1, Ehhadh.
Expression of gene products regulated by
PP in a PPARa-dependent manner were also
examined in liver tissues. Protein
levels of ACO, a rate limiting enzyme in
fatty acid b-oxidation pathway, and
Cyp4a were higher in untreated dwarf
mice and PP-treated wild type mice
compared to untreated wild type mice.
In addition, PPARa mRNA and protein
levels were constitutively upregulated
in dwarf mice likely contributing to the
observed increased expression of PPARa
gene targets. ACO and Cyp4a were also
upregulated in Ames dwarfs while Cyp4a
was increased in GHR-KO mice when
compared to corresponding wild type
mice. Differences in the expression of
genes and proteins involved in protein
folding (chaperonins, chaperones) and
cardiovascular disease were also
observed in dwarf mice. Overall, the
results indicated that some of the
beneficial effects associated with the
dwarf phenotype are likely due to the
constitutive activation of PPARa and
PPARa-regulated genes.
A second
body of evidence has been contributed by
Andrzej Bartke’s laboratory. These
investigators have systematically
evaluated PPAR expression levels in
several tissues of calorie-restricted
and ad libitum fed growth hormone
receptor/binding protein knock out (GHR-KO)
and wild type mice. The GHR-KO mice
exhibit GH resistance, reduced plasma
IGF-1, enhanced insulin sensitivity and
a 40% increase in life span. Liver
PPARa and PPARg mRNA and protein levels
were elevated as were retinoid X
receptor (RXR) mRNA levels in GHR-KO
mice compared to wild type mice.
Calorie restriction further enhanced
PPARa expression in the GHR-KO mice.
Differences in PPAR actions due to
altered expression levels likely
contribute to the enhanced insulin
sensitivity of GHR-KO mice. Tissue
specific differences were identified
when skeletal muscle was examined,
another target tissue of insulin. PPARg
and PPARa protein levels were reduced in
KO muscle while mRNA levels did not
differ from wild type mice. However, CR
reduced PPARa and PPARg mRNA and PPARa
protein in both KO and wild type mice.
Muscle RXR gene expression followed a
pattern similar to PPAR. In heart
tissue, the expression of PPARa and
PPARg did not differ between genotypes
but CR increased PPARa expression in GHR-KO
mice. These data suggest that the role
of PPARs in fatty acid metabolism and
the response to CR is tissue-specific
and dependent upon appropriate growth
factor signaling.
An
additional piece of the puzzle was
recently provided by work also from the
Bartke lab (PNAS, 2006). They
demonstrated that CR does not extend
lifespan in animals exhibiting GH-resistance
(GHR-KO) suggesting that the GH receptor
or GH receptor-dependent signaling
pathway is required for CR’s longevity
benefits in mammals. The lack of a life
extension benefit of CR in GHR-KO mice
was associated with a failure of CR to
further increase insulin sensitivity in
these mice.
Although
both GHR-KO and Ames (or Snell) dwarfs
are long-living and both exhibit reduced
IGF-1/insulin signaling, differences
between GHR-KO mice and these dwarf mice
exist. Dwarf mice subjected to CR live
longer than ad libitum fed dwarf mice.
In addition, it was shown that the Ames
mutation and CR affect a number of
overlapping genes and that additive
effects of the dwarf mutation and CR on
life span arise from additive effects on
the level of expression of some genes
and their independent effects on other
genes. Genes included in this category
that were under control of PGC-1 include
CAR, PXR and PPARa. And, while Ames
dwarf mice have reduced cholesterol,
triglyceride and free fatty acid levels,
GHR-KO mice only exhibit a significant
reduction in total plasma cholesterol
compared to wild type control mice.
Calorie restriction raised FFA in plasma
and muscle of wild type and KO mice
while lowering triglycerides in both
plasma and muscle. Cholesterol was
reduced in wild type mice following CR,
a finding consistent with previous
studies of calorie restriction. In
addition, dwarf mice lack prolactin (GHR-KO
– high prolactin) and thyroid hormone
levels are lower leading to differences
in reproductive ability, body
composition, oxidative stress resistance
and end-of-life pathology between these
two mutant strains.
Given the
role of PPARa in mediating
transcriptional responses of CR,
PGC-1-regulated PPARa may be one common
pathway that operates in dwarf mutants
and mice subjected to CR. Several
questions come to mind regarding the
role of PGC-1, downstream nuclear
receptor activation, calorie restriction
and the growth factor signaling
pathways. Will the beneficial actions of
calorie restriction ultimately be linked
to an active GH receptor in other mouse
strains/mammals? What is the role of
growth hormone? In C. elegans some of
the life extending mutations appear to
be acting through mechanisms similar to
CR but expression of insulin-like
pathways genes are not related. In
contrast, evidence in Drosophila
demonstrates great similarities between
genes activated by CR and those in the
insulin-like pathway. Regarding species
other than mammals, will the life
extending benefit of calorie restriction
be an underlying theme connected to a
functional growth factor pathway? Does
the type of restriction applied (every
other day feeding, protein restriction,
methionine restriction) make a
difference regarding PGC-1 and nuclear
hormone activation? Does CR represent a
stressor, thus upregulating a variety of
defense pathways via PGC-1? Is there
cross-species conservation of the
age-dependent declines in
PGC-1-regulated genes? Is the
upregulation of PPARa primarily for
increasing energy production needed for
tissue repair, optimal expression of
repair genes or increased protective
mechanisms?
Generation
and utilization of a number of nuclear
receptor mutants in combination with
calorie restriction will address many of
these questions. A clearer
understanding of CR will effectively aid
in the search for pharmacological agents
or lifestyle changes that may slow aging
and/or attenuate development or
progression of age-related disease.
Exploitation of nuclear hormone receptor
pharmacology in the design of CR
mimetics may be fertile ground in the
search for mediators of the aging
process.
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IN MEMORIAM
VINCENT J. CRISTOFALO, PhD
1933 - 2006
 Vincent
J. Cristofalo died on May 8, 2006; he was 73.
During his more than four decades of research in
cell and molecular biology he produced 138
peer-reviewed papers, 109 reviews, edited 15
books, and published 161 abstracts.
Additionally, he was series editor for 6
publications. He was a Professor at the Wistar
Institute and the University of Pennsylvania for
many years. After retiring, he moved to
Alleghany University to found the Center for
Gerontological Research, he also served as the
Vice-Provost of that Institution from 1994 -
1999. In 1999, he became President and CEO of
the Lankenau Institute for Medical Research
(Wynnewood, PA). He was also appointed a
Professor of Pathology, Anatomy and Cell Biology
by Thomas Jefferson University in 2000. The
recipient of many grants and awards, he was the
Principal Investigator on the longest running
program project grant in NIA history. He was
well traveled and presented scholarly work to
many different scientific organizations.
Additionally, he was President of the
Gerontological Society of America in 1990 and
President of the American Federation for Aging
Research (1996-1998). He was a member of
numerous editorial boards for a variety of
Gerontology journals and was the editor of "The
Journals of Gerontology: Biological Sciences"
from 1988-1991.
Throughout his
career, Dr. Cristofalo examined many aspects of
human cell growth and immortalization. Perhaps,
most famously, he extensively analyzed the
cessation of growth that occurs in cultures of
non-cancer cells that are maintained under
conditions that promote continuous growth. This
limit in the capacity of cell cultures to divide
has frequently been compared to aging in intact
organisms. Cristofalo explored many aspects of
this phenomenon at all levels of cellular
organization. He studied the effects of
oxygen, antioxidants and growth factors on
replicative capacity and posed questions about
the genetics and evolutionary involvement in
aging. He examined the stability of various
cellular components in young and aged cells and
identified many of the changes that occur during
proliferative senescence. Much of the work that
described both genetic and stochastic aspects of
cellular senescence was pioneered in his
laboratory.
In 1998, he and
others published what was possibly his most
controversial work entitled "Relationship
Between Donor Age and the Replicative Life Span
of Human Cells in Culture." In it, they
presented results that demonstrated that cells
from elderly people did not necessarily exhibit
diminished proliferative capacity as compared
with cells of young people. This paper,
published in The Proceedings of the National
Academy of Sciences, and several subsequent
works challenged not only a fundamental tenet of
the cellular senescence model but also redefined
cellular senescence on the basis of whether it
occurred in organisms or under culture
conditions as two distinct phenomena. In
another pivotal work, he discovered that the
b-gal assay commonly used as a specific marker
to link cell senescence with aging in vivo was
not actually specific to senescent cells and can
even be detected in cultures of cancer cells
that become confluent or that are treated with
oxidants. During the final years of his career
he examined changes in gene expression of
regulatory mechanisms that contributed to
senescence. He also collaborated on a project
that examined telomere length and proliferative
capacity in a cloned cow. Of course, his
greatest legacy is the dozens of students and
post-docs that studied in his lab and who
continue to contribute to understanding the
phenomenon of aging.
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