U.S. patent application number 13/984157 was filed with the patent office on 2014-03-06 for methods of treating mitochondrial dysfunction.
This patent application is currently assigned to ECOLE POLYTECHNIQUE FEDERALE DE LAUSANNE (EPFL). The applicant listed for this patent is Carlos Canto Alvarez, Johan Auwerx, Peter Bai, Riekelt Houtkooper, Laurent Mouchiroud. Invention is credited to Carlos Canto Alvarez, Johan Auwerx, Peter Bai, Riekelt Houtkooper, Laurent Mouchiroud.
Application Number | 20140065099 13/984157 |
Document ID | / |
Family ID | 46456943 |
Filed Date | 2014-03-06 |
United States Patent
Application |
20140065099 |
Kind Code |
A1 |
Alvarez; Carlos Canto ; et
al. |
March 6, 2014 |
Methods of Treating Mitochondrial Dysfunction
Abstract
The present invention provides methods of treating various
disorders associated with mitochondrial dysfunction, including but
not limited to metabolic disorders, neurodegenerative diseases,
chronic inflammatory diseases, and diseases of aging.
Inventors: |
Alvarez; Carlos Canto;
(Ecublens, CH) ; Bai; Peter; (Basel-Debrecen,
HU) ; Houtkooper; Riekelt; (Amsterdam, NL) ;
Auwerx; Johan; (Buchillon, CH) ; Mouchiroud;
Laurent; (Lausanne, CH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Alvarez; Carlos Canto
Bai; Peter
Houtkooper; Riekelt
Auwerx; Johan
Mouchiroud; Laurent |
Ecublens
Basel-Debrecen
Amsterdam
Buchillon
Lausanne |
|
CH
HU
NL
CH
CH |
|
|
Assignee: |
ECOLE POLYTECHNIQUE FEDERALE DE
LAUSANNE (EPFL)
Lausanne
CH
|
Family ID: |
46456943 |
Appl. No.: |
13/984157 |
Filed: |
February 15, 2012 |
PCT Filed: |
February 15, 2012 |
PCT NO: |
PCT/IB12/01146 |
371 Date: |
November 20, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61443052 |
Feb 15, 2011 |
|
|
|
61446303 |
Feb 24, 2011 |
|
|
|
Current U.S.
Class: |
424/85.2 ;
435/375; 514/43 |
Current CPC
Class: |
C12N 15/1137 20130101;
A61K 31/473 20130101; A61K 31/706 20130101; A61K 45/06 20130101;
A61K 31/7064 20130101; A61K 31/502 20130101; A61K 31/7088 20130101;
C12N 2310/14 20130101; A61K 31/405 20130101; A61K 31/05
20130101 |
Class at
Publication: |
424/85.2 ;
514/43; 435/375 |
International
Class: |
A61K 31/706 20060101
A61K031/706; A61K 45/06 20060101 A61K045/06 |
Claims
1. A method of treating a disorder associated with mitochondrial
dysfunction comprising administering to subject suffering from or
susceptible to developing a metabolic disorder one or more
compounds that increases intracellular nicotinamide adenine
dinucleotide (NAD.sup.+) in an amount sufficient to activate SIRT1
or SIRT3.
2. A method of promoting oxidative metabolism comprising
administering to subject suffering from or susceptible to
developing a metabolic disorder one or more compounds that
increases intracellular nicotinamide adenine dinucleotide
(NAD.sup.+) in an amount sufficient to activate SIRT1 or SIRT3.
3. The method of claim 1, wherein said disorder associated with
mitochondrial dysfunction is a metabolic disorder, a
neurodegenerative disease, an aging related disorder or a chronic
inflammatory disease.
4. The method of claim 3, wherein the metabolic disorder is obesity
or type II diabetes.
5. A method of treating cancer comprising administering to subject
suffering from or susceptible to developing a cancer a PARP
inhibitor and a NAD+ booster, a PARP inhibitor and an AMPK agonist,
or an AMPK agonist and a NAD+ booster.
6. The method according to claim 1, wherein the compound is a NAD
booster, a PARP-1 inhibitor, an AMPK activator or combination
thereof.
7. The method of claim 5, wherein the PARP inhibitor is PJ34, TIQ,
TES-500, TES-501, BSI-202, Iniparib, AZD2281, Olaparib, ABT-888,
Veliparib, AG014699, CEP 9722, or MK 4827.
8. The method of claim 5, wherein the PARP-1 inhibitor is a nucleic
acid that inhibits PARP-1 expression or activity.
9. The method of claim 5, wherein the NAD booster is tryptophan,
nicotinamide riboside (NR), niacin, nicotinic acid (NA),
nicotinamide (NAM), N-formylkynurenine, quinolinic acid,
nictotinamide riboside kinase (NRK) or nicotinamide mononucleotide
(NMN).
10. The method of claim 5, wherein the AMPK agonist is
5-aminoimidazole-4-carboxamide-1-b-D-ribosicie, PT-1, A-769662
(Abbott), Adiponectin, Leptin, Ghrelin, Cannabinoids, alpha-lipoic
acid, Interleukin-6 (IL-6), Resveratrol, Quercetin, Metformin,
Berberine, Curcumine, Epigallocatechin-3-gallate (green tea),
Thiazolidinediones, such as rosiglitazone and pioglitazone or
Dinitrophenol (DNP).
11. A method of increasing the concentration of NAD.sup.+ within
the mitochondria comprising contacting mitrocondria with
nicotinamide riboside (NR).
12. A method of activating mitochondrial sirtuin comprising
contacting mitochondria with nicotinamide riboside (NR).
13. The method of claim 12, wherein the sirtuin is SIRT3, SIRT4 or
SIRT5.
Description
RELATED APPLICATIONS
[0001] This application claims the benefit of provisional
applications U.S. Ser. No. 61/443,052 filed Feb. 15, 2011 and U.S.
Ser. No. 61/446,303 filed Feb. 24, 2011, the contents which are
each herein incorporated by reference in their entirety.
FIELD OF THE INVENTION
[0002] The present invention relates generally to methods of
increasing intracellular NAD.sup.+ for the treatment of various
mitochondrial disorders, including but not limited to metabolic
disorders, neurodegenerative diseases, and chronic inflammatory
diseases, and diseases associated with aging.
BACKGROUND OF THE INVENTION
[0003] Mitochondria are cellular organelles present in most
eukaryotic cells. One of their primary functions is oxidative
phosphorylation, a process through which energy derived from
metabolism of fuels like glucose or fatty acids is converted to
ATP, which is then used to drive various energy-requiring
biosynthetic reactions and other metabolic activities. Mitochondria
have their own genomes, separate from nuclear DNA, comprising rings
of DNA with about 16,000 base pairs in human cells. Each
mitochondrion may have multiple copies of its genome, and
individual cells may have hundreds of mitochondria.
[0004] Mitochondrial dysfunction contributes to various disease
states. Some mitochondrial diseases are due to mutations or
deletions in the mitochondrial genome. Mitochondria divide and
proliferate with a faster turnover rate than their host cells, and
their replication is under control of the nuclear genome. If a
threshold proportion of mitochondria in a cell is defective, and if
a threshold proportion of such cells within a tissue have defective
mitochondria, symptoms of tissue or organ dysfunction can result.
Practically any tissue can be affected, and a large variety of
symptoms may be present, depending on the extent to which different
tissues are involved.
[0005] In addition to congenital disorders involving inherited
defective mitochondria, acquired mitochondrial dysfunction
contributes to diseases, particularly neurodegenerative disorders
associated with aging like Parkinson's, Alzheimer's, Huntington's
Diseases. The incidence of somatic mutations in mitochondrial DNA
rises exponentially with age; diminished respiratory chain activity
is found universally in aging people. Mitochondrial dysfunction is
also implicated in excitotoxic neuronal injury, such as that
associated with seizures or ischemia. Other disorders associated
with mitochondrial dysfunction include chronic inflammatory
disorders and metabolic disorders
[0006] While a number of drugs have been developed over the years
to treat the various mitochondrial dysfunction, these drugs can
often have side effects or are effective only for a limited time
period. Thus a need exists for therapapetic strategies for treating
mitochondrial dysfunction
SUMMARY OF THE INVENTION
[0007] The invention features methods of treating disorders
associated with mitochondrial dysfunction by administering to
subject suffering from or susceptible to developing a metabolic
disorder one or more compounds that increases intracellular
nicotinamide adenine dinucleotide (NAD.sup.+) in an amount
sufficient to activate SIRT1 or SIRT3.
[0008] Also included in the invention are methods of promoting
oxidative metabolism by administering to subject suffering from or
susceptible to developing a metabolic disorder one or more
compounds that increases intracellular nicotinamide adenine
dinucleotide (NAD.sup.+) in an amount sufficient to activate SIRT1
or SIRT3.
[0009] In another aspect the invention provide a method of
increasing the concentration of NAD.sup.+ within the mitochondria
by contacting mitrocondria with nicotinamide riboside (NR).
[0010] Further included in the invention is a method of activating
mitochondrial sirtulin by contacting mitochondria with nicotinamide
riboside (NR). The sirtulin is SIRT3, SIRT4 or SIRT5.
[0011] Disorders associated with mitochondrial dysfunction is a
metabolic disorder, a neurodegenerative disease, a chronic
inflammatory disease, or an aging related disorder. For example,
the metabolic disorder is obesity or type II diabetes.
[0012] The compound is a NAD booster, a PARP-1 inhibitor, an AMPK
activator or combination thereof.
[0013] In another aspect the invention provides methods of treating
cancer comprising administering to subject suffering from or
susceptible to developing a cancer a PARP inhibitor and a NAD+
booster, a PARP inhibitor and an AMPK agonist, or an AMPK agonist
and a NAD+ booster.
[0014] PARP inhibitors include for example, PJ34, TIQ, TES-500,
TES-501, BSI-202, Iniparib, AZD2281, Olaparib, ABT-888, Veliparib,
AG014699, CEP 9722, or MK 4827. Alternatively, a PARP-1 inhibitor
is a nucleic acid that inhibits PARP-1 expression or activity.
[0015] A NAD booster includes for example tryptophan, nicotinamide
riboside (NR), niacin, nicotinic acid (NA), nicotinamide (NAM),
N-formylkynurenine, quinolinic acid, nictotinamide riboside kinase
(NRK) or nicotinamide mononucleotide (NMN).
[0016] An AMPK agonist is
5-aminoimidazole-4-carboxamide-1-b-D-riboside, PT-1, A-769662
(Abbott), Adiponectin, Leptin, Ghrelin, Cannabinoids, alpha-lipoic
acid, Interleukin-6 (IL-6), Resveratrol, Quercetin, Metformin,
Berberine, Curcumine, Epigallocatechin-3-gallate (green tea),
Thiazolidinediones, such as rosiglitazone and pioglitazone or
Dinitrophenol (DNP).
[0017] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention pertains.
Although methods and materials similar or equivalent to those
described herein can be used in the practice of the present
invention, suitable methods and materials are described below. All
publications, patent applications, patents, and other references
mentioned herein are expressly incorporated by reference in their
entirety. In cases of conflict, the present specification,
including definitions, will control. In addition, the materials,
methods, and examples described herein are illustrative only and
are not intended to be limiting.
[0018] Other features and advantages of the invention will be
apparent from and encompassed by the following detailed description
and claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] FIG. 1. Increased energy expenditure and adaptive
thermogenesis in PARP-1.sup.-/- mice. (A) PARP-1.sup.+/+ and
.sup.-/- male mice (n=8/9) were weighed weekly to analyze body
weight evolution. (B) Total white adipose tissue (WAT) was weighed
upon autopsy. (C) Average weekly food consumption throughout the
study. (D) O2 consumption and (E) respiratory-/- quotient (RQ) of
PARP-1.sup.+/+ and male mice (n=9/9) were measured by indirect
calorimetry. (F) Thermogenic capacity was determined upon acute
exposure of PARP-1.sup.+/+ and .sup.-/- mice to 4.degree. C. for
the indicated times (n=6/5 males). (G) Oral glucose tolerance test
was performed (n=5/5 males) and the area under curve (AUC) is shown
on the top right, expressed in arbitrary units. (H-J) Peripheral
and hepatic insulin responsiveness of PARP-1.sup.+/+ and
PARP-1.sup.-/- mice was assed by euglycemic-hyperinsulinemic clamp.
(H) Glucose infusion rates (GIR), (I) hepatic glucose production
(HGP) and (J) glucose uptake in different tissues are all shown as
mean+/-SEM. Throughout the figure, * indicates statistical
difference vs. PARP-1.sup.+/+ mice at p<0.05.
[0020] FIG. 2. PARP-1.sup.-/- mice are protected against high fat
feeding-induced metabolic abnormalities. (A). PARP-1 autoPARylation
band (arrowhead) was analyzed in 100 Rg of total protein extract
from gastrocnemius muscle obtained from 16 week-old wild-type
C57Bl/6J mice fed ad libitum or fasted for 24 h before sacrifice at
8:00 am. 50 .mu.g of protein were used to determine PARP-1 and
tubulin protein expression. (B) C57Bl/6J mice were fed chow or a
high-fat diet for 12 weeks and gastrocnemius muscle were analyzed
as described in (A). (C) PARP-1.sup.+/+ and -/- male mice.
(n=10/10) were fed chow (represented as circles) or a high-fat diet
(represented as squares) from the age of eight weeks onwards and
body weight was monitored weekly. (D) The total WAT mass,
individual WAT depots, and organ weights were determined upon
autopsy (E--epididymal, SC--subcutaneous, P--perirenal). (E) An
oral glucose tolerance test and (F) an intraperitoneal insulin
tolerance test were performed on high-fat fed PARP-1.sup.+/+ and
.sup.-/- male mice at 12 weeks of age (n=10/10). The area under
curve (AUC) of the oral glucose tolerance test is shown on the
top-right side of panel E, expressed in arbitrary units. In the
figure, * indicates statistical difference vs. PARP-1.sup.+/+ mice
at p<0.05.
[0021] FIG. 3. Increased mitochondrial activity in brown adipose
tissue (BAT) and gastrocnemius muscle of PARP-1.sup.-/- mice. (A)
BAT and after of PARP-1.sup.+/+ and .sup.-/- was photographed
weighed autopsy and mice (11.5 months of age, n=8/9 males). BAT
content (relative to total body weight), is shown at the bottom of
the image. (B) BAT mitochondrial DNA (mtDNA) was quantified by
qPCR. (C) mRNA levels of the indicated genes were determined by
RT-qPCR in the BAT. (D) Transmission electron micrographs of
representative BAT sections show increased mitochondrial content in
PARP-1.sup.-/- mice. (E-F) 25 .mu.g of total protein extracts from
(E) BAT or (F) gastrocnemius muscles of PARP-1.sup.+/+ and .sup.-/-
mice were used to analyze the abundance of mitochondrial complexes.
(G) SDH staining of sections from the gastrocnemius and soleus
muscles of PARP-1.sup.+/+ and .sup.-/- (H) mRNA levels of the
indicated genes were measured by RT-qPCR in gastrocnemius muscle.
Throughout the figure, white bars represent PARP-1.sup.+/+ mice,
while black bars represent PARP1.sup.-/- mice. * indicates
statistical difference vs. PARP-1.sup.+/+ mice at p<0.05.
[0022] FIG. 4. The absence of PARP-1 raises NAD.sup.+ levels and
activates SIRT1 (A) Protein PARylation was determined by anti-PAR
staining on formalin-fixed 7 .mu.m BAT and muscle tissue sections
of PARP-1.sup.+/+ and .sup.-/- mice. The white bar is equivalent to
10 .mu.m. (B) NAD.sup.+ and (C) NAM levels in BAT and muscle were
determined by mass spectrometry. (D-E) PARP-1, SIRT1 and actin (as
loading control) protein content on (D) BAT and (E) skeletal muscle
was determined by Western blotting using 100 .mu.g of total protein
lysate. PGC-1.alpha. and FOXO1 acetylation was examined by
immunoprecipitation. (F) Tubulin and acetylated-tubulin levels were
estimated in gastrocnemius muscle from PARP-1.sup.+/+ and .sup.-/-
mice. (G) The Ndufa9 subunit of mitochondrial complex I was
immunoprecipitated from 400 .mu.g of total protein from
gastrocnemius muscle and acetylation levels of the complex were
analyzed by western blotting. * indicates statistical difference
vs. PARP-1.sup.+/+ mice at p<0.05. Abbreviations can be found in
the text.
[0023] FIG. 5. PARP-1 knock-down promotes SIRT1 activity and
oxidative metabolism. (A-C) HEK293T cells were transfected with
either a scramble (as control) or a PARP-1 shRNA and
HA-PGC-1.alpha. for 48 h. (A) Total cell lysates were then obtained
to analyze PARP-1 protein and PARP-1 autoPARylation (arrowhead).
(B) Intracellular NAD+ levels was measured on total acid extracts.
(C) Total protein lysates were used to analyze PGC-1.alpha.
deacetylation in HA immunoprecipitates. (D-F) HEK293T cells were
transfected with either a pool of PARP-1 siRNAs, a pool of SIRT1
siRNAs, or different combinations of both using the corresponding
scramble siRNAs as control (-). Additionally, the cells were
simultaneously transfected with HA-PGC-1.alpha. for 48 h. Then, (D)
relative mitochondrial DNA content, (E) mRNA levels of the markers
indicated and (F) total O.sub.2 consumption were analyzed as
described. * indicates statistical difference vs. respective
control sh/siRNA-transfected cells at p<0.05.
[0024] FIG. 6. Pharmacological PARP-1 inhibition activates SIRT1
and enhances mitochondrial function in cultured cells. (A-C) C2C12
myotubes, which express FLAG-HA-PGC-1.alpha., were treated for 6
hrs with either PBS (as vehicle), H.sub.2O.sub.2 (500 .mu.M) or
H2O2 and PJ34 (1 .mu.M). Then, (A) total protein extracts were
obtained to test the markers indicated, (B) Intracellular NAD+ and
SIRT1 protein levels were measured and (C) PGC-1.alpha. acetylation
was tested in FLAG immunoprecipitates. Tubulin was measured on the
supernatants to ensure equal protein input. (D) C2C12 mytobues were
treated with PJ34 (1 mM) for the times indicated and acidic
extracts were evaluate NAD.sup.+ obtained tointracellular levels.
(E-F) C2C12 myotubes, which express FLAG-HA-PGC-1.alpha., were
treated for 24 h with PBS (as vehicle) or with the PARP inhibitor
PJ34 (1 .mu.M, unless otherwise stated). (E) PARP-1 protein and
PARP-1 autoPARylation (arrowhead) were then determined by Western
blotting and (F) intracellular NAD.sup.+ content and PGC-.alpha.
acetylation levels were measured. (G-I) C2C12 myotubes
differentiated for 48 h were infected with FLAG-HA-PGC-1.alpha. and
a control or a SIRT1 shRNA. 48 h later, myotubes were treated with
PJ34 for 24 hrs (unless otherwise stated). Then, (G) PGC-1.alpha.
acetylation levels were quantified by immunoprecipitation. 50 Rg of
total protein extracts were used to measure the other markers
indicated. (H) mRNA expression levels of selected genes were
quantified 48 hrs by RT-qPCR reactions; abbreviations are listed in
the text. (I) Cellular O.sub.2 consumption was measured 48 hrs
after PJ34 treatment as described. * indicates statistical vs.
vehicle-treated group at p<0.05.
[0025] FIG. 7. Pharmacological PARP-1 inhibition increases
NAD.sup.+ levels and phenocopies SIRT1 activation in vivo. C57Bl/6J
male mice received daily injections with PJ34 (2.times.10 mg/kg/day
i.p.) or saline (n=10/10) for 5 days before sacrifice; then (A-B)
Global PARylation, p-ACC and SIRT1 levels were determined by using
100 Rg of total protein extracts from (A) BAT and (B) gastrocnemius
muscle. The PARP-1 autoPARylation band is indicated by an
arrowhead. (C-D) NAD.sup.+ levels and PGC-1 acetylation levels were
determined in (C) BAT and (D) in muscle. To detect PGC-1
acetylation, 1 mg of BAT and 2 mg of protein from gastrocnemius
muscle were used to immunoprecipitate PGC-1.alpha. using 5 .mu.g of
antibody. Then acetyl-lysine levels were evaluated by western blot.
(E-F) mRNA expression levels of selected genes in (E) BAT and (F)
gastrocnemius muscle were quantified by RT-qPCR; abbreviations are
listed in the text. * indicates statistical vs. vehicle-treated
group at p<0.05.
[0026] FIG. 8. Increased spontaneous locomotor activity and energy
expenditure in the PARP-1.sup.-/- mice during night. (A) Oxygen
consumption was determined in PARP-1.sup.+/+ and .sup.-/- mice
(n=6/6) as described in the Materials and Methods. TEE-total energy
expenditure, REE-resting energy expenditure. (B) Spontaneous
activity was determined during indirect calorimetry in CLAMS using
PARP-1.sup.+/+ and (n=9/9). Asterisks indicate significant
difference between cohorts, where * p<0.05.
[0027] FIG. 9. Gene expression of different metabolic genes and
PARP1 in metabolic tissues. (A) mRNA expression levels of selected
genes were quantified by RT-qPCR reactions in the liver of
PARP-1.sup.+/+ and .sup.-/- mice (n=9/9); abbreviations are listed
in the text. Asterisks indicate significant difference between
cohorts, where * p<0.05; *** p<0.001. (B) mRNA expression of
PARP-1 were quantified in different metabolic tissues of C57Bl/6J
male mice (n=5). Asterisks indicate significant difference between
the respective tissue and liver, where *** p<0.001.
[0028] FIG. 10. Gene expression pattern of the different members of
the PARP family in the BAT and gastrocnemius muscle RT-qPCR
reactions were performed on cDNA populations from the BAT (A) and
the gastrocnemius muscle (B) of PARP-1.sup.+/+ and .sup.-/- male
mice (n=7/5). Asterisks indicate significant difference between
cohorts, where * p<0.05; *** p<0.001.
[0029] FIG. 11. Assessment of mitochondrial function and protein
levels in PARP-1.sup.+/+ and -/- MEFs. In PARP-1.sup.+/+ and
.sup.-/- primary MEFs (n=3/3) oxygen consumption (A), mitochondrial
DNA content (B), mitochondrial membrane potential (C), mRNA
expression, (D) phospho-ACC, SIRT1 and PARP-1 protein levels (E)
was determined. Abbreviations are listed in the text. Asterisks
indicate significant difference between cohorts, where * p<0.05,
** p<0.01.
[0030] FIG. 12. Assessment of mitochondrial function
upon-/-pharmacological PARP inhibition in C2C12 cells and SIRT1
MEFs. (A) Promoter occupancy of PGC-1.alpha. was quantified after
PJ34 treatment on the PDK4 and UCP-3 promoters (1 .mu.M, 48 h)
(n=3/3). Mitochondrial DNA (B), mitochondrial membrane potential
(C) was characterized in differentiated C2C12 myofibers after PJ34
treatment (1 .mu.M, 48 h) (n=3/3). (D) Substrate contribution to
biological oxidation was determined as described in the Materials
and Methods. (E-G) PGC-1 acetylation, ACC phosphorylation, SIRT1
protein levels (E), expression of mRNAs encoding for mitochondrial
proteins (F) and O2 consumption (G) were determined in
SIRT1.sup.+/+ and MEF.sup.-/- cells.
[0031] FIG. 13. PARP-2 regulates oxidative metabolism by acting as
a transcriptional repressor of SIRT1. (A) PARP-2 protein and mRNA
levels were analyzed in C2C12 myotubes carrying stably transfected
scramble or PARP-2 shRNA. (B) NAD.sup.+ content was evaluated in
C2C12 myotubes treated with PJ34 (24 hrs, 1 mM) or carrying a
stable transfection of a scramble or a PARP-2 shRNA. H.sub.2O.sub.2
treatment was performed for 1 hr. (C) Total protein extracts from
C2C12 mytotubes treated as in (B) were used to test total
PARylation (D) Scramble or PARP-2 shRNA were stably transfected in
C2C12 myotubes that were infected with FLAG-PGC-1. After 48 hr,
total protein extracts were obtained and used for FLAG
immunoprecipitation and to test the markers indicated. (E) SIRT1
mRNA levels were analyzed in C2C12 myotubes carrying a stable
transfection with either scramble or a PARP-2 siRNA. (F) The
activity of nested deletions of the SIRT1 promoter was measured
after PARP-2 depletion in C2C12 cells. (G) The presence of PARP-2
on the SIRT1 (-1--91) and K19 promoter was assessed in C2C12 cells
by ChIP assays. (H-I) O.sub.2 consumption (H) and mRNA levels of
the markers indicated (I) were measured in C2C12 myotubes carrying
a stable transfection with either a scramble (-) or a PARP-2 (+)
shRNA and infected with adenovirus encoding for either a scramble
(-) or a SIRT1 (+) shRNA. Unless otherwise indicated, white bars
represent scramble shRNA transfected myotubes and black bars
represent PARP-2 shRNA transfected myotubes. All results are
expressed as mean.+-.SD. * indicates statistical difference vs.
PARP-2.sup.+/+ mice at p<0.05.
[0032] FIG. 14. General physiologic characteristics of
PARP-2.sup.-/- mice. (A) PARP-2.sup.+/+ and male mice (n=15/13)
were weighed weekly and (B) food consumption was measured. (C-E)
PARP-2.sup.+/+ and male mice on a chow diet (n=6/6, age of 3
months) were subjected to indirect calorimetry, where (C) locomotor
activity, (D) O.sub.2 consumption and (E) RER were determined. (F)
Fed and fasted blood glucose levels. * indicates statistical
difference vs. PARP-2.sup.+/+ mice at p<0.05.
[0033] FIG. 15. PARP-2.sup.-/- muscles have higher SIRT1 activity,
mitochondrial content and oxidative profile. (A) PARylation and
PARP-2 levels in gastrocnemius muscle were determined by western
blot. PARP-2 levels were determined in nuclear extracts, and
histone 1 (H1) was used as loading (B) NAD.sup.+ control levels in
gastrocnemius muscle of 4-months oldPARP-2+/+ and .sup.-/- male
mice (n=4 and 8, respectively) were determined by HPLC/MS (C) SIRT1
mRNA and protein levels were determined in total muscle mRNA or
protein extracts. (D) PGC-1.alpha. and (E) FOXO1 acetylation lysine
levels were examined after immunoprecipitation. Quantifications are
shown on top of the respective images. (F) Gene expression of the
indicated genes in the gastrocnemius muscle of PARP-2.sup.+/+ and
.sup.-/- mice was evaluated by RT-qPCR. (G) Quantification of
mitochondrial DNA by qPCR(H) Transmission electron micrographs and
(I) SDH staining of representative gastrocnemius muscle sections
show increased mitochondrial content (PARP-2.sup.+/+ and .sup.-/-
male mice n=15 and 13, respectively; age of 7 months). Scale bar in
(I)=100 m (J) Endurance treadmill test was performed as described.
White bars represent PARP-2.sup.+/+ mice, while black bars
represent PARP-2.sup.-/- mice. * indicates statistical difference
vs. PARP-2.sup.+/+ mice at p<0.05.
[0034] FIG. 16. PARP-2.sup.-/- mice display higher mitochondrial
content in liver. (A) mRNA expression in livers from PARP-2+/+
analysis and male (n=16/13, respectively; 6 months of age) mice fed
a chow diet. (B) Relative liver mitochondrial DNA (mtDNA) content
was estimated by RT-qPCR. (C) Transmission electron microscopic
images of liver sections demonstrate higher mitochondrial number in
PARP-2.sup.-/- mice. (D) Total intrahepatic NAD.sup.+ content was
measured by HPLC/MS. (E) Total liver protein extracts were used to
evaluate SIRT1 protein levels and immunoprecipitate PGC-1 to
examine PGC-1 acetylation levels. (F) Liver triglyceride content
was estimated after methanol/chloroform lipid extraction as
described. White bars represent PARP-2.sup.+/+ mice, while black
bars represent PARP-2.sup.-/- mice. * indicates statistical
difference vs. PARP-2.sup.+/+ mice at p<0.05.
[0035] FIG. 17. PARP-2.sup.-/- mice are protected against
diet-induced body weight gain and insulin resistance. (A) 6 month
old PARP-2+/+ and .sup.-/- male mice (n=7 and 9, respectively) fed
on high fat diet were weighed weekly (B) Food intake was monitored
during high-fat feeding. (C) Body fat mass composition was
evaluated through EchoMRI. (D) The weight of the tissues indicated
was determined upon autopsy at the end of the high-fat feeding
period. (E) VO.sub.2 and (F) spontaneous activity was determined by
indirect calorimetry. Quantification of the mean values during
light and dark phases are shown. (G) mRNA expression levels in
gastrocnemius muscles from PARP-2.sup.+/+ and .sup.-/- mice after
12 weeks of high-fat diet was determined by qRT-PCR (H) Glucose
excursion after an intraperitoneal insulin tolerance test. White
bars and circles represent PARP-2.sup.+/+ mice, while black bars
and circles represent PARP-2.sup.-/- mice. * indicates statistical
difference vs. PARP-2.sup.+/+ mice at p<0.05.
[0036] FIG. 18. Pancreatic abnormalities render PARP-2.sup.-/- mice
glucose intolerant after high-fat feeding. (A) Plasma glucose
levels during an intraperitoneal glucose tolerance test (IPGTT) in
9-month old PARP-2.sup.+/+ and .sup.-/- male mice (n=7 and 9,
respectively) fed a high fat diet for 12 weeks. The area under the
curve of the glucose curves is shown at the right. (B) Insulin
levels during the first hour of the IPGTT in (A). (C) Comparison of
total pancreas weight between PARP-2.sup.+/+ and .sup.-/- mice on
chow and high-fat diet. (D) Pancreas from PARP-2.sup.+/+ and
.sup.-/- mice after high-fat diet were stained for insulin (scale
bar=50 Rm) and (E) Mean islet size was quantified. (F) Total
insulin content in pancreas was measured as described. (G) Gene
expression in the pancreas of PARP-2.sup.+/+ and .sup.-/- mice was
measured by RT-qPCR. (H) Pancreatic total protein extracts were
used to test the abundance of SIRT1, and subunits from the
respiratory complexes I and III. FOXO1 was also immunoprecipitated
to determine relative FOXO1 acetylation levels. Through the figure,
white bars and circles represent PARP-2.sup.+/+ mice, while black
bars and circles represent PARP-2.sup.-/- mice. * indicates
statistical difference vs. PARP-2.sup.+/+ mice at p<0.05.
[0037] FIG. 19. PARP-2 influences SIRT1 activity by directly
regulating the SIRT1 promoter. (A) Total and mitochondrial
NAD.sup.+ was determined as described in Experimental procedures in
C2C12 cells transduced with either scramble (white bars) or a
PARP-2 (black bars) shRNA. (B) PARP-2 and SIRT1 were
immunoprecipitated from C2C12 cells and blotted for the markers
indicated. (C) ChIP assay was performed in HEK293T cells and the
interaction of PARP-2 with the SIRT1-91 bp promoter region (black
bars) or the K19 promoter (white bars) was evaluated by qPCR. (D)
Alignment of the SIRT1 promoter of different vertebrate species was
performed using the ClustalW software. The green field indicates
the murine -1--91 region, where PARP-2 interacts. * indicates
statistical difference between the PARP-2 IP and the unspecific
antibody binding at P<0.05.
[0038] FIG. 20. PARP-2 deletion does not lead to the accumulation
of DNA damage and does not influence SIRT2 and SIRT3 activity in
muscle. (A) Representative image of the TUNEL reaction in the
gastrocnemius muscle of young (3 months of age) and old (11 months
of age) PARP-2.sup.+/+ and .sup.-/- male mice (n=3/3/4/3; young
PARP-2.sup.+/+/young PARP-2.sup.-/-/old PARP-2.sup.+/+/old
PARP-2.sup.-/-) to determine the amount of DNA strand breaks. The
bar represents 1 m. Arrows represent TUNEL-positive nuclei
indicative of DNA damage. (B) The acetylation status of tubulin, a
SIRT2 target, was evaluated using specific antibodies, while the
activity of SIRT3 activity was evaluated by the acetylation status
of Ndufa9 (37 kDa) immunoprecipitates.
[0039] FIG. 21. PARP-2 deletion does not have a major impact on BAT
gene expression and function. (A) SIRT1 protein levels were
detected in the BAT of PARP-2.sup.+/+ and .sup.-/- male mice by
Western blotting. (B) BAT mRNA expression pattern was determined in
PARP-2.sup.+/+ and male mice (n=16/13) by RT-qPCR. (C).
PARP-2.sup.+/+ and .sup.-/- mice (n=6/6) were exposed to cold
(4.degree. C.), as described in Experimental procedures. White bars
and circles represent PARP-2.sup.+/+ mice and black bars or circles
represent PARP-2.sup.-/- mice. All results are expressed as
mean.+-.SD.
[0040] FIG. 22. PARP-2.sup.-/- livers display reduced lipid
accumulation, in the absence of changes in SIRT2 and SIRT3 activity
or gluconeogenic potential. (A) The acetylation of SIRT2 and SIRT3
targets (tubulin and Ndufa9, respectively) were determined by the
use of specific antibodies (acetyl-tubulin) or by
immunoprecipitation (Ndufa9). (B) Liver morphology and lipid
content was assessed by hematoxilin-eosin (HE) and Oil-Red O (ORO)
stainings. The bar represents 10 .mu.m. (C) Gluconeogenesis was
assessed by intraperitoneal pyruvate PARP-2+/+--tolerance test in
(white bar and circles) and/--(black bar and circles) male mice
(n=10/9) as described in materials and methods. The area under
curve (AUC) is shown at the right of the panel * indicates
statistical difference PARP-2.sup.+/+ vs. PARP-2.sup.-/- mice at
p<0.05.
[0041] FIG. 23. PARP-2.sup.-/- livers are protected from high-fat
diet-induced lipid accumulation. (A) Morphology and lipid
accumulation the liver of PARP-2.sup.+/+ and .sup.-/- male mice
(n=16/13) after 12 weeks of high-fat diet was visualized with
hematoxilin-eosin (HE) and Oil Red-O staining. The bar represents
20 .mu.m. (B) Triglyceride quantity was determined after lipid
extraction from PARP-2.sup.+/+ (white bar) or PARP-2.sup.-/- (black
bar) livers as described in the methods. * indicates statistical
difference PARP-2.sup.+/+ HFD vs. PARP-2.sup.-/- HFD mice at
p<0.05.
[0042] FIG. 24. The pancreas of PARP-2.sup.-/-, but not
PARP-1.sup.-/- mice, is hypofunctional upon high-fat feeding. (A)
Total NAD+ was determined from the pancreas of PARP-2.sup.+/+
(white bar) and .sup.-/- male mice (black bar) (n=7/5). (B) The
absence of an interaction between pancreatic PARP-2 and FOXO-1 was
evidenced by immunprecipitation experiments. (C) The pancreas of
PARP-1.sup.+/+ and .sup.-/- male mice (n=3/3) were stained for
insulin (bar=50 .mu.m). (D) Insulin content in the pancreas from
PARP-1.sup.+/+ (white bar) or -/- (black bar) mice was determined
by ELISA.
[0043] FIG. 25. PARP inhibitors are a useful tool to increase
intracellular NAD.sup.+ content (A) C2C12 myotubes were treated for
24 hrs with different PARP inhibitors at the concentrations
indicated. (B) C2C12 myotubes were treated with PBS (Veh), PJ34 (1
mM) or TES501 at the concentrations indicated. (C) C2C12 myotubes
were treated with PJ34 or TES501 for the times indicated. *
indicates statistical difference vs. untreated or vehicle treated
cells at p<0.05.
[0044] FIG. 26. Nicotinamide Riboside supplementation increases
NAD.sup.+ content and sirtuin activity in cultured mammalian cells.
(A) C2C12 myotubes, Hepa1.6 and HEK293T cells were treated with
nicotinamide riboside (NR) for 24 hrs and acidic extracts were
obtained to measure total NAD.sup.+ intracellular content. (B)
GPR109A-expressing Chem-4 cells were loaded with 3 .mu.M Fura-2
acetoxymethyl ester derivative (Fura-2/AM) for 30 min at 37.degree.
C. Then, cells were washed with Hank's balanced salt solution and
calcium flux in response to nicotinic acid (NA; as positive
control), NR and nicotinamide mononucleotide (NMN) at the
concentrations indicated was determined as indicated in methods.
(C) C2C12 myotubes, Hepa1.6 and HEK293T cells were treated with
either PBS (as Vehicle) or 0.5 mM of NR, NMN or NA for 24 hrs. Then
total NAD.sup.+ intracellular content was determined as in (A). (D)
C57Bl/6J mice were fed with chow containing vehicle (water) or
either NR, NMN or NA at 400 mg/kg/day (n=8 mice per group). After
one week, NAD.sup.+ content was determined in liver and quadriceps
muscle. (E) HEK293T cells were treated with NR (0.5 mM, black bars)
or vehicle (white bars) for 4 hrs. Then, cells were harvested and
mitochondria were isolated for NAD.sup.+ measurement. (F) C57Bl/6J
mice were fed with chow containing vehicle (water) or NR at 400
mg/kg/day (n=8 mice per group). After one week, mitochondria were
isolated from their livers to measure NAD.sup.+ content. (G)
HEK293T cells were treated with either PBS (as Vehicle) or 0.5 mM
of NR for 24 hrs. Then mRNA and protein was extracted to measure
Nampt levels by RT-qPCR and western blot, respectively. (H) HEK293T
cells were treated with either PBS (as Vehicle) or 0.5 mM of NR for
24 hrs. Then protein homogenates were obtained to test global
PARylation and PARP-1 levels. Throughout the figure, all values are
presented as mean+/-SD. * indicates statistical significant
difference vs. respective vehicle group at P<0.05. Unless
otherwise stated, the vehicle groups are represented by white bars,
and NR groups are represented by black bars.
[0045] FIG. 27. Nicotinamide Riboside supplementation increases
sirtuin activity in cultured mammalian cells. (A) HEK293T cells
were transfected with a pool of either scramble siRNAs or SIRT1
siRNAs. After 24 hrs, cells were treated with vehicle (PBS) or NR
at the concentrations indicated, and, after an additional 24 hrs,
total protein extracts were obtained. FOXO1 acetylation was tested
after FOXO1 immunoprecipitation (IP) from 500 .mu.g of protein,
while tubulin and SIRT1 levels were evaluated in the supernatant of
the IP. (B) HEK293T cells were transfected with a pool of either
scramble siRNAs, FOXO1 siRNAs or SIRT1 siRNAs. After 24 hrs, cells
were treated with NR (0.5 mM; black bars) or vehicle (PBS; white
bars) for additional 24 hrs. Then total mRNA was extracted and the
mRNA expression levels of the markers indicated was evaluated by
qRT-PCR. (C) HEK293T cells were transfected with a pool of either
scramble siRNAs, FOXO1 siRNAs or SIRT1 siRNAs. After 24 hrs, cells
were treated with NR (0.5 mM; black bars) or vehicle (PBS; white
bars) for additional 24 hrs. Then acidic extracts were obtained to
measure intracellular NAD.sup.+ levels. (D-E) HEK293T cells were
treated with NR (0.5 mM) or vehicle (PBS) for 24 hrs and total
protein extracts were obtained to measure (D) Ndufa9 or (E) SOD2
acetylation after IP. The extracts were also used to measure SOD2
activity (E, bottom panel). (F-G). SIRT3.sup.+/+ and SIRT3.sup.-/-
mouse embryonic fibroblasts (MEFs) were treated with NR (0.5 mM) or
vehicle (PBS) for 24 hrs and either (F) total extracts to test SOD2
acetylation were obtained or (G) acidic extracts were used to
measure intracellular NAD.sup.+ content. Throughout the figure, all
values are presented as mean+/-SD. * indicates statistical
significant difference vs. respective vehicle group at P<0.05.
Unless otherwise stated, the vehicle groups are represented by
white bars, and NR groups are represented by black bars.
[0046] FIG. 28. NR supplementation prevents diet-induced obesity by
enhancing energy expenditure and reduces cholesterol levels.
10-week-old C57Bl/6J mice were fed with either chow (CD) or high
fat diet (HFD) mixed with either water (as vehicle) or NR (400
mg/kg/day) (n=10 mice per group). (A) Body weight evolution was
monitored during 12 weeks. (B) Body composition was evaluated after
8 weeks of diet through Echo-MRI. (C-E) Food intake, activity and
VO.sub.2 were evaluated using indirect calorimetry. (F-G) Blood
glucose and insulin levels were measured in animals fed with their
respective diets for 16 weeks after a 6 hr fast. (H-I) After 10
weeks on their respective diets (CD=squares; RFD=circles) an
intraperitoneal glucose tolerance test was performed in mice that
were fasted overnight. At the indicated times blood samples were
obtained to evaluate either (H) glucose or (I) insulin levels.
Areas under the curve are shown at the top-right of the respective
panels (J) Hyperinsulinemic-euglycemic clamps were performed on
either CD or CD-NR mice (4 weeks of treatment). Glucose infusion
rates (GIR) and muscle glucose uptake were calculated after the
test. (K) Serum levels of total cholesterol were measured in
animals fed with their respective diets for 16 weeks, after a 6 hr
fast. Throughout the figure, white represent the vehicle group and
black represent the NR-supplemented mice. All values are presented
as mean+/-SD. * indicates statistical significant difference vs.
respective vehicle treated group.
[0047] FIG. 29. NR enhances skeletal muscle and BAT oxidative
function. 10-week-old C57Bl/6J mice were fed a high fat diet (HFD)
mixed with either water (as vehicle; white bars and circles) or NR
(400 mg/kg/day; black bars and circles) (n=10 mice per group). (A)
An endurance exercise test was performed using a treadmill in mice
fed with either HFD or HFD-NR for 12 weeks. (B) A cold-test was
performed in mice fed with either HFD or HFD-NR for 9 weeks. The
area over the curve (AOC) is shown on the top right of the graph.
(D) Electron microscopy of the BAT was used to analyze
mitochondrial content and morphology. The size and cristae content
of mitochondria was quantified as specified in methods. Throughout
the figure, all values are shown as mean+/-SD. * indicates
statistical significant difference vs. vehicle supplemented group
at P<0.05.
[0048] FIG. 30. Chronic NR supplementation increases plasma and
intracellular NAD.sup.+ content in a tissue-specific manner.
Tissues from C57Bl/6J mice were collected after 16 weeks of HFD
supplemented with either water (as vehicle; white bars) or N.sup.R
(400 mg/kg/day; black bars). (A) NAD.sup.+ levels were measured in
acidic extracts obtained from different tissues. (B) NADH and NAM
levels were measured in gastrocnemius muscle. (C) Quadriceps muscle
protein homogenates were obtained to test global PARylation, PARP-1
and Nampt protein levels. (D) Total mRNA was isolated from
quadriceps muscles and the mRNA levels of the markers indicated
were measured by RT-qPCR. Throughout the figure, all values are
expressed as mean+/-SD. * indicates statistical significant
difference vs. respective vehicle treated group.
[0049] FIG. 31. NR stimulates sirtuin activity in vivo and enhances
mitochondrial gene expression. Tissues from C57Bl/6J mice were
collected after 16 weeks of HFD supplemented with either water (as
vehicle; white bars) or NR (400 mg/kg/day; black bars). (A) Total
protein extracts were obtained from quadriceps muscle and brain
indicated to evaluate the acetylation levels of FOXO1 and SOD2
through immunoprecipitation assays, using 1 and 0.5 mg of protein,
respectively. (B) Total mRNA from quadriceps muscle and brain was
extracted to measure the abundance of the markers indicated by
RT-qPCR. (C) Mitochondrial DNA content was measured in DNA
extracted from quadriceps muscle and brain. The results are
expressed a mitochondrial copy number relative to genomic DNA (D)
The abundance of mitochondrial marker proteins in 20 .mu.g of
protein from total quadriceps muscle and brain lysates. Throughout
the figure, all values are shown as mean+/-SD. * indicates
statistical significant difference vs. vehicle supplemented group
at P<0.05.
[0050] FIG. 32. Schematic representation of the different actions
of NR in metabolic homeostasis. The scheme summarizes the
hypothesis by which NR supplementation would increase NAD.sup.+
content in key metabolic tissues, leading to SIRT1 and SIRT3
activation and the deacetylation and modulation of the activity of
key metabolic regulators. This model does not rule out the
participation of additional mechanisms of action for NR to achieve
its beneficial effects. Abbreviations can be found in the text and
enzymes are indicated in italics.
[0051] FIG. 33. PARP activity and NAD+ in aged mammals and worms.
(A) Total protein PARylation was evaluated in liver and muscle of
young (6 months) and aged (24 months) C57BL/6J mice, and was
accompanied by (B) decreased NAD+ levels, and (C) PGC-1
hyperacetylation. (D) Aged C. elegans displayed higher total
protein PARylation levels, which were largely attenuated in pme-1
mutants. (E) Aging decreased worm NAD+, in both wildtype and in
pme-1 mutant worms, with a higher level of NAD+ in the pme-1 mutant
during aging. Two-way ANOVA revealed significant difference with
age (p<0.008) and genotype (p=0.02). (F) pme-1 mutant worms
accumulated less of the aging pigment lipofuscin compared to wild
type worms. Bar graphs are expressed as mean.+-.SEM, *
p.ltoreq.0.05; ** p.ltoreq.0.01; *** p.ltoreq.0.001.
[0052] FIG. 34. Longevity in C. elegans with pme-1 mutation or PARP
inhibition. (A) pme-1 (ok988) mutant worms displayed 29.4% mean
lifespan extension. (B) PARP inhibition by AZD2281 (100 nM) or
ABT-888 (100 nM), extended lifespan by 22.9% and 15% respectively,
(C) in a pme-1-dependent manner. (D) pme-1 mutation and PARP
inhibition increased NAD+ levels in C. elegans at day 4 of
adulthood. (E) PARP inhibition by AZD2281 (100 nM) does not extend
lifespan in the sir-2.1(ok434) mutant. Bar graphs are expressed as
mean.+-.SEM, *p<0.05.
[0053] FIG. 35. PARP inhibition increases mitochondrial function
and ROS defense. (A) AZD2281 decreased the accumulation of the
aging pigment lipofuscin. (B) Oxygen consumption was increased in
day 3 adult worms after AZD2281 (AZD). (C) Quantitative RT-PCR
analysis of AZD2281-treated worms at day 3 of adulthood shows
increased expression of genes involved in mitochondrial oxidative
metabolism. (D-E) The effects of AZD on mitochondrial content and
morphology in body wall muscle. Stars represent nuclei, insets show
higher magnification of a small section of the image, marked by the
dashed rectangle. (F) AZD2281 decreased ROS, as measured by
mitoSOX, accompanied by an increase in sod-3::GFP. (G-H)
Quantitative-RT-PCR for oxidative stress regulators in vehicle- and
AZD2281-treated worms. (I) AZD2281 fails to extend lifespan in
daf-16(mu86) mutant worms. Bar graphs are expressed as mean.+-.SEM,
* p.ltoreq.0.05; ** p.ltoreq.0.01; *** p.ltoreq.0.001.
[0054] FIG. 36. Supplementation of C. elegans with the NAD+
precursor NR mimics the metabolic and lifespan effects of PARP
mutation or inhibition. Supplementation of NAD.sup.+ precursors NR
(500 RM) in wild type N2 worms increases (A) NAD.sup.+ and (B-C)
lifespan in a sir-2-dependent manner. (D-E) The effects of NR on
mitochondrial content and morphology in body wall muscle. Stars
represent nuclei, insets show higher magnification of a small
section of the image, marked by the dashed rectangle. (F) Oxygen
consumption was increased in day 3 adult worms after NR. (G-H)
Quantitative-RT-PCR of the expression of oxidative stress-related
genes in wild-type and NR-treated worms. (I) NR effects on lifespan
are daf-16-dependent. (J) Scheme summarizing how NAD.sup.+
precursors and PARP inhibitors increase lifespan through activation
of sir-2.1. Bar graphs are expressed as mean.+-.SEM, *p<0.05;
**p<0.01.
[0055] FIG. 37. Lifespan analyses with different concentrations of
PARP inhibitors. (A-B) Worm lifespan was measured after treatment
with PARP inhibitors AZD2281 (A) or ABT-888 (B). See Table S1 for
statistics.
[0056] FIG. 38. Lifespan analyses with different concentrations of
NAD+ precursors. (A-B) Worm lifespan was measured after treatment
with NAD+ precursors NR (A) or NAM (B). See Table S1 for
statistics.
[0057] FIG. 39. The NAD precursor NAM increases NAD+ levels,
lifespan, mitochondrial function and ROS defense. (A) NAM increased
NAD+ levels in C. elegans at day 4 of adulthood. (B) NAM increased
worm lifespan by 18%. (C) Oxygen consumption was increased in day 3
adult worms after NAM. (D) Quantitative-RT-PCR of NAM-treated worms
revealed a trend for increased cts-1 expression, and expression of
sod-3 was increased whereas daf-16 was unchanged (E) NAM increased
mitochondrial content in body wall muscle. Bar graphs are expressed
as mean.+-.SEM, *p<0.05; ** p<0.01.
[0058] FIG. 40. Is a schematic illustrating how one can modulate of
energy metabolism by impacting on NAD.sup.+ levels. This scheme
illustrates that in addition to inhibiting NAD+ consuption (e.g.
through PARP inhibitors) or changing the ratio between NAD+/NADH
(e.g. through AMPK activators) also providing more NAD+ precursors
(e.g. Nicotinamide Riboside (NR), Nicotinic acid (NA), Nicotimamide
(NAM), Nicotinamide mononucleotide (NMN), Tryptophan) could be used
to increase NAD+ levels.
DETAILED DESCRIPTION OF THE INVENTION
[0059] The invention is based upon the discovery of new pathways to
regulate intracellelluar NAD.sup.+. The inventors have shown that a
decrease in poly(ADP-ribose) polymerase-1 (PARP-1) activity induces
intracellular NAD.sup.+, SIRT1 and SIRT3 activity and that a
decrease on poly(ADP-ribose) polymerase-2 (PARP-2) activity induces
SIRT1 and SIRT3 activity. More specifically, it was discovered that
inhibition of PARP-1 and PARP-2 promotes oxidative metabolism and
oxidative stress defence. Similar effects can be obtained through
boosting NAD+ levels by providing NAD+ precursors such as
nictinamide riboside or nicotinic acid. Furthermore, the inventors
have also discovered that NAD+ is an aging biomarker. Specifically,
the inventors have shown that NAD+ levels and sirtuin activity are
reduced in aged mice and C. elegans. Restoration of NAD+ levels, by
genetic or pharmacological reduction of PARP-1 PARP-2 or by
increasing the supply of NAD+ precursors prevents age-associated
metabolic decline and extends lifespan in a sir-2.1-dependent
fashion.
[0060] Regulation of intracellular NAD+ levels are useful in
treating or alliveating a symptom of various disorders in which
aberrant (i.e., increase or decrease) mitochondrial function is
involved. For example, regulation of intracellular NAD+ levels is
useful in treating or alleviating a symptom of mitochondrial
disorders which include diseases with inherited and/or acquired
mitochondrial dysfunction, such as Charcot-Marie-Tooth disease,
Type 2A2, Mitochondrial Encephalopathy Lactic Acidosis and Stroke
(MELAS), Leigh Syndrome, Barth Syndrome, Leber's optic neuropathy,
fatty acid oxidation disorders, inherited forms of deafness and
blindness, metabolic abnormalities induced by exposure to toxic
chemicals and/or drugs (e.g. cisplatin induced deafness, gentamycin
induced deafness). In addition, the methods of the invention are
also useful at treating or alliveating a symptom of metabolic
disorders, neurodegenerative disorders, aging related disorders or
chronic inflammatory disorders, all characterized by mitochondrial
dysfunction.
[0061] Intracellular NAD+ levels control the activity of the type
III deacetylase SIRT1 (Lin et al., 2000), allowing it to act as a
metabolic sensor and fine-tune transcriptional programs to drive
the utilization of different energetic substrates (Gerhart-Hines et
al., 2007; Rodgers et al., 2005). Overexpression studies have
revealed how enhancing the activity of SIRT1 or of its orthologs
promotes longevity in lower eukaryotes (reviewed by (Canto and
Auwerx, 2009)) and protects against high-fat diet (HFD)-induced
metabolic disease in mice (Banks et al., 2008; Pfluger et al.,
2008), which in turn may also indirectly sustain a more healthy
ageing process. These attractive properties of SIRT1 activation
have spurred a quest to identify SIRT1 "activators" that could be
used pharmacologically in situations of metabolic stress and
damage. Most of the previous attempts to pharmacologically activate
SIRT1 have relied on the discovery of direct small molecule SIRT1
agonists. This strategy has identified compounds, like resveratrol
or SRT1720 (Borra et al., 2005; Howitz et al., 2003; Kaeberlein et
al., 2005; Milne et al., 2007; Pacholec et al., 2010), whose
ability to directly interact and activate SIRT1 is still under
debate (Borra et al., 2005; Canto et al., 2010; Dai et al., 2010;
Kaeberlein et al., 2005; Pacholec et al., 2010; Urn et al., 2009).
Consequently, there is a strong interest to develop alternative
strategies to activate SIRT1. Given the NAD.sup.+-sensing abilities
of SIRT1, another potential way to activate it would be by
increasing intracellular NAD.sup.+ levels. The present invention is
based on the hypothesis that SIRT1 can be activated by specific
inhibition of other cellular NAD+-consuming activities.
[0062] Poly(ADP-ribose) polymerase (PARP)-1 constitutes one of the
major NAD.sup.+ consumers in the cell (Schraufstatter et al., 1986;
Shieh et al., 1998). PARP-1 is activated upon binding to damaged or
abnormal DNA (Durkacz et al., 1980; Kun et al., 2002), and
catalyzes the formation of poly(ADP-ribose) polymers (PAR) onto
different acceptor proteins, including PARP-1 itself
(auto-PARylation), using NAD.sup.+ as substrate (Adamietz, 1987;
Burkle, 2005; Chambon et al., 1963). To test the influence of
PARP-1 on SIRT1 activity and on metabolic homeostasis we used both
a genetic strategy, exploiting PARP-1 deficient (PARP-1.sup.-/-)
mouse (Menissier-de Murcia et al., 1997) and cellular models, and a
pharmacological approach, to inhibit PARP-1 activity. The combined
results as described herein of these complimentary studies
demonstrate how a reduction or ablation of PARP-1 activity
increases NAD.sup.+ levels and SIRT1 activity, which, in turn,
promotes mitochondrial content and function, culminating in a solid
protection against metabolic disease.
[0063] PARP-2 has a structurally similar catalytic domain (amino
acids 202-593) as PARP-1 (Oliver et al., 2004). Accordingly, we
also evaluated the effect of PARP-2 inhibition on intracellular
NAD.sup.+ levels and global metabolism in cells or organs. The
potential relevance of PARP-2 for NAD.sup.+ homeostasis, which
would impact on SIRT1 activity and global metabolism, prompted us
hence to fully examine the metabolic phenotype of germline
PARP-2.sup.-/- mice. The data shown herein demonstrates that the
absence of PARP-2 activates SIRT1 and promotes mitochondrial
biogenesis in muscle. However, our data also reveals that the
absence of PARP-2 leads to pancreatic failure upon high-fat
feeding, underscoring the possibility of developing drugs that
selectively inhibit specific PARP proteins for metabolic
indications.
[0064] Accordingly the invention features methods of promoting
oxidative metabolism and treating, alleviating a symptom or
delaying the onset of a disorder associated with aberrant
mitochondrial function by administering to a subject a compound
that increases intracellular nicotinamide adenine dinucleotide
(NAD.sup.+) in an amount sufficient to activate SIRT1 or SIRT3.
Also included in the invention are methods of treating, alleviating
a symptom or delaying the onset cancer by one or more compounds
that increases intracellular nicotinamide adenine dinucleotide
(NAD.sup.+) in an amount sufficient to activate SIRT1 or SIRT3. The
subject is suffering from or susceptible to developing the
disorder.
[0065] The invention further provides methods of increasing
concentration of NAD.sup.+ within the mitocondria by contacting
mitochondria with nicotinomide riboside. In another aspect, the
invention provides a method of activating mitrocondrial sirtuins.
Mitochondrial sirtuins include for example SIRT3, SIRT4 and
SIRT5.
[0066] Compounds that increase NAD.sup.+ include inhibitors of the
poly (ADP-ribose) polymerase (PARPs) family of proteins, NAD+
boosters and AMPK agonists. The compounds can be administered alone
or in combination.
[0067] Members of the PARPs family of protein include PARP-1,
PARP-2, PARP-3, PARP-4, PARP-5a, PARP5b, PARP-6, PARP-7, PARP-8,
PARP-9, PARP-10, PARP-12, PARP-13, PARP-14, PARP-15, and PARP-16.
Preferably, the compound is a PARP-1 inhibitor.
[0068] A PARP-1 inhibitor is a compound that decreases expression
or activity of PARP-1. A decrease in PARP-1 expression or activity
is defined by a reduction of a biological function of the PARP-1
protein. A PARP-1 biological function includes for example, the
catalysis of lipid molecules between phospholipid membranes or the
transfer of lipid from high density lipoproteins (HDL) to low
density lipoproteins (LDL). PARP-1 expression is measured by
detecting a PARP-1 transcript or protein or by measuring PARylation
activity. PARP-1 inhibitors are known in the art or are identified
using methods described herein. For example, a PARP-1 inhibitor is
identified by detecting an increase of intracellular NAD.sup.+.
Intracellular NAD.sup.+ is detected by methods known in the art
such the methods disclosed herein.
[0069] The PARP-1 inhibitor is for example an antisense PARP-1
nucleic acid, a PARP-1 specific short-interfering RNA, or a PARP
specific ribozyme.
[0070] By the term "siRNA" is meant a double stranded RNA molecule
which prevents translation of a target mRNA. Standard techniques of
introducing siRNA into a cell are used, including those in which
DNA is a template from which an siRNA RNA is transcribed. The siRNA
includes a sense PARP-1 nucleic acid sequence, an anti-sense PARP-1
nucleic acid sequence or both. Optionally, the siRNA is constructed
such that a single transcript has both the sense and complementary
antisense sequences from the target gene, e.g., a hairpin.
[0071] Binding of the siRNA to a PARP-1 transcript in the target
cell results in a reduction in PARP-1 production by the cell. The
length of the oligonucleotide is at least 10 nucleotides and may be
as long as the naturally-occurring PARP-1 transcript. Preferably,
the oligonucleotide is 19-25 nucleotides in length. Most
preferably, the oligonucleotide is less than 75, 50, 25 nucleotides
in length.
[0072] Exemplary PARP-1 inhibitors, which inhibit NAD+ consumption,
also include small molecule inhibitors such as PJ34, TIQ, TES-500,
TES-501, BSI-202 or Iniparib, AZD2281 or Olaparib, ABT-888 or
Veliparib, AG014699, CEP 9722 MK 4827. Other PARP-1 inhibitors are
known in the art.
[0073] Other examples of molecules that can raise NAD+ levels,
independently of PARP inhibition, are compounds that induce NAD+
synthesis (i.e. NAD boosters), such as tryptophan, nicotinamide
riboside (NR), niacin, nicotinic acid (NA), nicotinamide (NAM),
N-formylkynurenine, Quionlinic acid, nictotinamide riboside kinase
(NRK) or nicotinamide mononucleotide (NMN).
[0074] Exemplary compounds that also induce NAD+ levels,
independent of the stimulation of NAD+ synthesis or the inhibition
of NAD+ usage, include small molecule activators of AMP activated
kinase (AMPK), such as
5-aminoimidazole-4-carboxamide-1-b-D-riboside, PT-1, A-769662
(Abbott), Adiponectin, Leptin, Ghrelin, Cannabinoids, alpha-lipoic
acid, Interleukin-6 (IL-6), Resveratrol, Quercetin, Metformin,
Berberine, Curcumine, Epigallocatechin-3-gallate (green tea),
Thiazolidinediones, such as rosiglitazone and pioglitazone or
Dinitrophenol (DNP).
[0075] The present invention provides for both prophylactic and
therapeutic methods of treating a subject at risk of (or
susceptible to) a disorder or having a disorder associated with
aberrant (e.g., insufficient) metabolism. As used herein, the term
"treatment" is defined as the application or administration of a
therapeutic agent to a patient, or application or administration of
a therapeutic agent to an isolated tissue or cell line from a
patient, who has a disease, a symptom of disease or a
predisposition toward a disease, with the purpose to cure, heal,
alleviate, relieve, alter, remedy, ameliorate, improve or affect
the disease, the symptoms of disease or the predisposition toward
disease.
[0076] Oxidative metabolism is promoted by exposing, e.g.,
contacting a tissue or cell with a compound that increases that
increases intracellular nicotinamide adenine dinucleotide
(NAD.sup.+) in an amount sufficient to activate SIRT1. By promoting
oxidative metabolism is meant an increase in oxygen consumption
compared to a tissue or cell that has not been in contact with
compound. Tissues or cells are directly contacted with compound.
Alternatively, the compound is administered systemically. The
compound is administered in an amount sufficient to increase (e.g.,
activate) SIRT1 or SIRT3. Oxidative metabolism is measured by known
in the art, such as by the methods described herein.
[0077] The methods are useful to treat, alleviate the symptoms of,
or delay the onset of a disorder associated with aberrant
mitochondrial function. Disorders associated with aberrant
mitochondrial function include for example metabolic disorders,
neurodegenerative disorders aging related disorders and chronic
inflammatory disorders. Mitochondrial disorders include also
diseases with inherited and/or acquired mitochondrial dysfunction,
such as Charcot-Marie-Tooth disease, Type 2A2, Mitochondrial
Encephalopathy Lactic Acidosis and Stroke (MELAS), Leigh syndrome,
Barth syndrome, Leber's optic neuropathy, Fatty acid oxidation
disorders, Inherited forms of deafness and blindness, metabolic
abnormalities induced by exposure to toxic chemicals and/or drugs
(e.g. cisplatin induced deafness).
[0078] Metabolic disorders include for example, type II diabetes,
obesity, hyperglycemia, glucose intolerance, insulin resistance
(i.e., hyperinsulinemia, metabolic syndrome, syndrome X),
hypercholesterolemia, hypertension, hyperlipoproteinemia,
hyperlipidemia (e.g., dyslipidemia), hypertriglylceridemia,
cardiovascular disease, atherosclerosis, peripheral vascular
disease, kidney disease, ketoacidosis, thrombotic disorders,
nephropathy, diabetic neuropathy, diabetic retinopathy, sexual
dysfunction, dermatopathy, dyspepsia, hypoglycemia, cancer or
edema.
[0079] Neurodegenerative disorders include diseases such as
Dementia, Alzheimer's disease, Parkinson's disease, and
Huntington's disease.
[0080] Chronic inflammatory diseases include disease such as celiac
disease, vasculitis, lupus, chronic obstructive pulmonary disease
(COPD), irritable bowel disease, atherosclerosis, arthritis, and
psoriasis.
[0081] Aging related disorders includes disease such as cancer,
dementia, ardiovascular disease, such as arteriosclerosis,
hypertension, diabetes mellitus (type I or type II) arthritis,
cataracts, Alzheimer's disease and osteoporosis.
[0082] The subject is suffering from or a susceptible to developing
a metabolic disorder. Subjects suffering from or at risk of
developing a metabolic disorder are identified by methods known in
the art. For example diabetes is diagnosed by for example by
measuring fasting blood glucose levels or insulin or by glucose
tolerance test. Normal adult glucose levels are 60-126 mg/dl.
Normal insulin levels are 7 mU/mL.+-.3mU. Hypertension is diagnosed
by a blood pressure consistently at or above 140/90. Cardiovascular
disease is diagnosed by measuring cholesterol levels. For example,
LDL cholesterol above 137 or total cholesterol above 200 is
indicative of cardiovascular disease. Hyperglycemia is diagnosed by
a blood glucose level higher than 10 mmol/l (180 mg/dl). Glucose
intolerance is diagnosed by a two-hour glucose levels of 140 to 199
mg per dL (7.8 to 11.0 mmol) on the 75-g oral glucose tolerance
test. Insulin resistance is diagnosed by a fasting serum insulin
level of greater than approximately 60 pmol/L. Hypoglycemia is
diagnosed by a blood glucose level lower than 2.8 to 3.0 mmol/L (50
to 54 mg/dl). Obesity is diagnosed for example, by body mass index.
Body mass index (BMI) is measured (kg/m.sup.2(or
lb/in.sup.2.times.704.5)). Alternatively, waist circumference
(estimates fat distribution), waist-to-hip ratio (estimates fat
distribution), skinfold thickness (if measured at several sites,
estimates fat distribution), or bioimpedance (based on principle
that lean mass conducts current better than fat mass (i.e., fat
mass impedes current), estimates % fat) is measured. The parameters
for normal, overweight, or obese individuals is as follows:
Underweight: BMI<18.5; Normal: BMI 18.5 to 24.9; Overweight:
BMI=25 to 29.9. Overweight individuals are characterized as having
a waist circumference of >94 cm for men or >80 cm for women
and waist to hip ratios of .gtoreq.0.95 in men and .gtoreq.0.80 in
women. Obese individuals are characterized as having a BMI of 30 to
34.9, being greater than 20% above "normal" weight for height,
having a body fat percentage >30% for women and 25% for men, and
having a waist circumference >102 cm (40 inches) for men or 88
cm (35 inches) for women. Individuals with severe or morbid obesity
are characterized as having a BMI of .gtoreq.35.
[0083] The methods described herein lead to a reduction in the
severity or the alleviation of one or more symptoms of the
metabolic disorder. Symptoms of diabetes include for example
elevated fasting blood glucose levels, blood pressure at or above
140/90 mm/Hg; abnormal blood fat levels, such as high-density
lipoproteins (HDL) less than or equal to 35 mg/dL, or triglycerides
greater than or equal to 250 mg/dL (mg/dL=milligrams of glucose per
deciliter of blood). Efficacy of treatment is determined in
association with any known method for diagnosing the metabolic
disorder. Alleviation of one or more symptoms of the metabolic
disorder indicates that the compound confers a clinical
benefit.
[0084] The compounds, e.g., PARP-1 inhibitors (also referred to
herein as "active compounds") of the invention, and derivatives,
fragments, analogs and homologs thereof, can be incorporated into
pharmaceutical compositions suitable for administration. Such
compositions typically comprise the peptide or mimetic, and a
pharmaceutically acceptable carrier. As used herein,
"pharmaceutically acceptable carrier" is intended to include any
and all solvents, dispersion media, coatings, antibacterial and
antifungal agents, isotonic and absorption delaying agents, and the
like, compatible with pharmaceutical administration. Suitable
carriers are described in the most recent edition of Remington's
Pharmaceutical Sciences, a standard reference text in the field,
which is incorporated herein by reference. Preferred examples of
such carriers or diluents include, but are not limited to, water,
saline, finger's solutions, dextrose solution, and 5% human serum
albumin. Liposomes and non-aqueous vehicles such as fixed oils may
also be used. The use of such media and agents for pharmaceutically
active substances is well known in the art. Except insofar as any
conventional media or agent is incompatible with the active
compound, use thereof in the compositions is contemplated.
Supplementary active compounds can also be incorporated into the
compositions.
[0085] Mitochondrial disorders are diagnosed for example in
combination with abnormalities of glucose and lipid homeostasis,
ketone bodies and abnormalities in acid/base balance and abnormal
levels of other metabolites in the blood.
[0086] Neurodegenerative disorders are diagnosed for example by
physical and neurological examination, family history,
Electroencephalograms (EEGs) MRI and CAT scans.
[0087] A pharmaceutical composition of the invention is formulated
to be compatible with its intended route of administration.
Examples of routes of administration include parenteral, e.g.,
intravenous, intradermal, subcutaneous, oral (e.g., inhalation),
transdermal (topical), transmucosal, and rectal administration.
Solutions or suspensions used for parenteral, intradermal, or
subcutaneous application can include the following components: a
sterile diluent such as water for injection, saline solution, fixed
oils, polyethylene glycols, glycerine, propylene glycol or other
synthetic solvents; antibacterial agents such as benzyl alcohol or
methyl parabens; antioxidants such as ascorbic acid or sodium
bisulfite; chelating agents such as ethylenediaminetetraacetic
acid; buffers such as acetates, citrates or phosphates, and agents
for the adjustment of tonicity such as sodium chloride or dextrose.
The pH can be adjusted with acids or bases, such as hydrochloric
acid or sodium hydroxide. The parenteral preparation can be
enclosed in ampoules, disposable syringes or multiple dose vials
made of glass or plastic.
[0088] Pharmaceutical compositions suitable for injectable use
include sterile aqueous solutions (where water soluble) or
dispersions and sterile powders for the extemporaneous preparation
of sterile injectable solutions or dispersion. For intravenous
administration, suitable carriers include physiological saline,
bacteriostatic water, Cremophor EL.TM. (BASF, Parsippany, N.J.) or
phosphate buffered saline (PBS). In all cases, the composition must
be sterile and should be fluid to the extent that easy
syringeability exists. It must be stable under the conditions of
manufacture and storage and must be preserved against the
contaminating action of microorganisms such as bacteria and fungi.
The carrier can be a solvent or dispersion medium containing, for
example, water, ethanol, polyol (for example, glycerol, propylene
glycol, and liquid polyethylene glycol, and the like), and suitable
mixtures thereof. The proper fluidity can be maintained, for
example, by the use of a coating such as lecithin, by the
maintenance of the required particle size in the case of dispersion
and by the use of surfactants. Prevention of the action of
microorganisms can be achieved by various antibacterial and
antifungal agents, for example, parabens, chlorobutanol, phenol,
ascorbic acid, thimerosal, and the like. In many cases, it will be
preferable to include isotonic agents, for example, sugars,
polyalcohols such as manitol, sorbitol, and sodium chloride in the
composition. Prolonged absorption of the injectable compositions
can be brought about by including in the composition an agent which
delays absorption, for example, aluminum monostearate and
gelatin.
[0089] Sterile injectable solutions can be prepared by
incorporating the active compound (e.g., a PARP-1 inhibitor) in the
required amount in an appropriate solvent with one or a combination
of ingredients enumerated above, as required, followed by filtered
sterilization. Generally, dispersions are prepared by incorporating
the active compound into a sterile vehicle that contains a basic
dispersion medium and the required other ingredients from those
enumerated above. In the case of sterile powders for the
preparation of sterile injectable solutions, methods of preparation
are vacuum drying and freeze-drying that yields a powder of the
active ingredient plus any additional desired ingredient from a
previously sterile-filtered solution thereof.
[0090] Oral compositions generally include an inert diluent or an
edible carrier. They can be enclosed in gelatin capsules or
compressed into tablets. For the purpose of oral therapeutic
administration, the active compound can be incorporated with
excipients and used in the form of tablets, troches, or capsules.
Oral compositions can also be prepared using a fluid carrier for
use as a mouthwash, wherein the compound in the fluid carrier is
applied orally and swished and expectorated or swallowed.
Pharmaceutically compatible binding agents, and/or adjuvant
materials can be included as part of the composition. The tablets,
pills, capsules, troches and the like can contain any of the
following ingredients, or compounds of a similar nature: a binder
such as microcrystalline cellulose, gum tragacanth or gelatin; an
excipient such as starch or lactose, a disintegrating agent such as
alginic acid, Primogel, or corn starch; a lubricant such as
magnesium stearate or Sterotes; a glidant such as colloidal silicon
dioxide; a sweetening agent such as sucrose or saccharin; or a
flavoring agent such as peppermint, methyl salicylate, or orange
flavoring.
[0091] For administration by inhalation, the compounds are
delivered in the form of an aerosol spray from pressured container
or dispenser which contains a suitable propellant, e.g., a gas such
as carbon dioxide, or a nebulizer.
[0092] Systemic administration can also be by transmucosal or
transdermal means. For transmucosal or transdermal administration,
penetrants appropriate to the barrier to be permeated are used in
the formulation. Such penetrants are generally known in the art,
and include, for example, for transmucosal administration,
detergents, bile salts, and fusidic acid derivatives. Transmucosal
administration can be accomplished through the use of nasal sprays
or suppositories. For transdermal administration, the active
compounds are formulated into ointments, salves, gels, or creams as
generally known in the art.
[0093] The compounds can also be prepared in the form of
suppositories (e.g., with conventional suppository bases such as
cocoa butter and other glycerides) or retention enemas for rectal
delivery.
[0094] In one embodiment, the active compounds are prepared with
carriers that will protect the compound against rapid elimination
from the body, such as a controlled release formulation, including
implants and microencapsulated delivery systems. Biodegradable,
biocompatible polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid; collagen, polyorthoesters, and
polylactic acid. Methods for preparation of such formulations will
be apparent to those skilled in the art. The materials can also be
obtained commercially from Alza Corporation and Nova
Pharmaceuticals, Inc. Liposomal suspensions (including liposomes
targeted to infected cells with monoclonal antibodies to viral
antigens) can also be used as pharmaceutically acceptable carriers.
These can be prepared according to methods known to those skilled
in the art, for example, as described in U.S. Pat. No. 4,522,811,
incorporated fully herein by reference.
[0095] It is especially advantageous to formulate oral or
parenteral compositions in dosage unit form for ease of
administration and uniformity of dosage. Dosage unit form as used
herein refers to physically discrete units suited as unitary
dosages for the subject to be treated; each unit containing a
predetermined quantity of active compound calculated to produce the
desired therapeutic effect in association with the required
pharmaceutical carrier. The specification for the dosage unit forms
of the invention are dictated by and directly dependent on the
unique characteristics of the active compound and the particular
therapeutic effect to be achieved.
[0096] The pharmaceutical compositions can be included in a
container, pack, or dispenser together with instructions for
administration.
EXAMPLES
Example 1
General Methods
[0097] Materials.
[0098] All chemicals, including PJ34 (Garcia et al., 2001), were
from Sigma-Aldrich unless stated otherwise.
[0099] Animal Experiments.
[0100] Male PARP-1.sup.+/+ and PARP-1.sup.-/- mice on a pure
C57Bl/6J background (Menissier-de Murcia et al., 1997) were used.
Mice were housed separately, had ad libitum access to water and
standard rodent chow (10 kcal % of fat, Safe, Augy, France) or to a
high calorie, high fat diet (60 kcal % of fat, Research Diets, New
Brunswick, N.J., USA), and were kept under a 12 h dark-light cycle.
In other animal experiments, 8 weeks-old male C57Bl/6J mice were
purchased from Charles River and powder chow (D12450B) and high fat
(D12492) diets were from Research Diets Inc (New Brunswick, N.J.,
USA). 80 ml of water per kg of powder CD were used to make food
pellets. 40 ml of water per kg of powder HFD were used to make food
pellets. For NR, NMN and NA supplemented diets, the appropriate
amount of these compounds was added to the water used to create the
pellets, taking into account the differences in the daily intake of
each diet. Mice were housed separately, had ad libitum access to
water and food and were kept under a 12 h dark-light cycle. Mice
were fed with homemade pellets from 10 weeks of age. To make the
pellets, the powder food was mixed with water (vehicle) or with NR.
All animal experiments were carried out according to local national
and EU ethical guidelines. To monitor body weight, mice were
weighed and the food consumption was measured each week on the same
day. In case of PJ34 treatment, mice received each 12 h (at 7:00
and 19:00) 10 mg/kg PJ34 by intraperitoneal injection for 5
continuous days. In all studies animals were killed (at 14:00)
either after CO.sub.2 inhalation or cervical dislocation after 6 h
of fasting (starting at 8:00), and tissues were collected and
processed as specified below. Oral glucose tolerance test,
intraperitoneal insulin tolerance test, free fatty acid (FFA) and
triglycerides were determined as described (Lagouge et al., 2006).
Plasma insulin and was determined in heparinized plasma samples
using specific ELISA kits (Mercodia). Blood samples were collected
in heparinized tubes and plasma was isolated after centrifugation.
Plasma parameters were measured using a Cobas c111 (Roche
Diagnostics). Thermoadaptation was performed as described (Lagouge
et al., 2006). We measured O.sub.2 consumption, CO.sub.2
production, and spontaneous locomotor activity in an open circuit
indirect calorimetry system (Sabre systems, Las Vegas, Nev., USA)
over 24-48 h as described (Dali-Youcef et al., 2007; Lagouge et
al., 2006; Watanabe et al., 2006). Energy expenditure was obtained
by using an energy equivalent of 20.1 J/ml O.sub.2. The respiratory
quotient was the ratio of CO.sub.2 production over O.sub.2
consumption. During actimetry, beamline crossings were summarized
each 15 minutes. The sum of beamline crosses for each 15 min period
were plotted against time and AUC was calculated for each mouse
that was average in each experimental cohort.
Euglycemic-hyperinsulinemic clamps were performed in PARP-1.sup.-/-
and .sup.+/+ male mice (n=4 mice per genotype; age=4 months)
exactly as previously described (Feige et al, 2008).
[0101] Homozygous PARP-2.sup.-/- and littermate PARP-2.sup.+/+ mice
(Menissier-de Murcia et al., 2003) and PARP-1.sup.-/- and
PARP-1.sup.+/+ mice (Menissier-de Murcia et al., 1997), on a mixed
C57BL/6J/SV129 background (87.5%/12.5%) background, from
heterozygous crossings were used. Mice were housed separately, had
ad libitum access to water and food, and were kept under a 12/12 h
dark-light cycle. Mice were selected for the study at the age of 4
weeks and were kept on chow diet (10 kcal % of fat, Safe, Augy,
France). For a part of the animals, food was changed by a high fat
diet (HFD, 60 kcal % of fat, Research Diets, New Brunswick, N.J.,
USA) at the age of 16 weeks. Each week on the same day mice were
weighed and the food-consumption was measured. O.sub.2 consumption
(VO.sub.2), CO.sub.2 production, respiratory exchange ratios (RER),
and actimetry were measured by CLAMS of Colombus
[0102] Instruments, total body fat was measured by echoMRI as
described (Lagouge et al., 2006; Watanabe et al., 2006). Endurance
test was performed as described in (Canto et al., 2009).
Intraperitoneal glucose and insulin tolerance tests (IPGTT and
IPITT, respectively) were performed as previously described (Feige
et al, 2008). The animals were killed after 6 h of fasting by
CO.sub.2 asphyxiation and tissues were collected. Total body fat
content was then examined at autopsy, by weighing the subcutaneous,
gonadal, mesenterial, retroperitoneal and BAT associated fat
depots. Liver triglyceride was determined after Folch extraction
using a commercial triglyceride kit (Roche). Pancreas and plasma
insulin content was determined from acidic extracts using a
commercial ELISA kit (Mercodia) (Champy et al., 2004).
[0103] Histology and Microscopy.
[0104] Haematoxylin-eosin (HE), Oil Red-O and
succinate-dehydrogenase (SDH) stainings were performed on 7 m
tissue sections as described (Lagouge et al., 2006; Picard et al.,
2002). Transmission electron microscopy (TEM) investigation was
performed on glutaraldehyde-fixed, osmium tetroxyde stained
ultrafine sections (Watanabe et al., 2006). PAR was detected in
tissues using a monoclonal anti-PAR antibody (Alexis) and
Mouse-on-mouse kit (Vector Laboratories) on 7 .mu.m formalin-fixed
tissues using as described in (Garcia et al., 2001). A specific
binding of the secondary antibody was controlled on sections where
the primary antibody was omitted. Mitochondrial size and cristae
content was analyzed as previously described in St-Pierre et al.
(St-Pierre, J., Lin, J., Krauss, S., Tarr, P. T., Yang, R.,
Newgard, C. B., and Spiegelman, B. M, Bioenergetic analysis of
peroxisome proliferator-activated receptor gamma coactivators
1alpha and 1beta (PGC-1alpha and PGC-1beta) in muscle cells. J Biol
Chem 278, 26597-26603 (2003)).
[0105] Cell Culture, Transfection, Adenoviral Infection and
Mitochondrial Characterization.
[0106] HEK293T, MEF and C2C12 cells were cultured in DMEM (4.5 g/l
glucose, 10% FCS). PARP-1.sup.+/+ and PARP-1.sup.-/- MEFs were
prepared as described in (Menissier-de Murcia et al., 1997).
SIRT1-/- and MEFs were kindly provided by Fred Alt (Chua et al.,
2005). SIRT3 MEFs were established according to standard techniques
from conditional SERT3.sup.-/- mice. (Picard, F., Gehin, M.,
Annicotte, J., Rocchi, S., Champy, M. F., O'Malley, B. W., Chambon,
P., and Auwerx, J. (2002). SRC-1 and TIF2 control energy balance
between white and brown adipose tissues. Cell 111, 931-941.)
Deletion of the SIRT3 gene was induced via infection with
adenovirus encoding for the Cre recombinase. HEK293T cells were
transfected using JetPei reagent (Polyplus Transfections, Illkirch,
France) according to the manufacturer's instructions. C2C12 cells
were differentiated in DMEM (4.5 g/L glucose, 2% horse serum) after
reaching confluency for 2 days, followed by 2 days of PJ34
treatment (10 .mu.M). PARP-1 shRNA constructs were described in
(Shah et al., 2005). Human PARP-1 and SIRT1 siRNAs were obtained
from Dharmacon (Thermo Scientific). The adenovirus encoding for
FLAG-HA-PGC-1.alpha., control and SIRT1 shRNAs were a kind gift
from Pere Puigserver and were used (MOI=100) in C2C12 myotubes as
described (Canto et al., 2009). Mitochondrial charge determination
was performed as described (Bai et al., 2001) and DNA strand breaks
were quantified by TUNEL assays according to the manufacturer's
instructions (Millipore).
[0107] Murine Hepa1.6 and HEK293T cells were maintained in 4.5 g/L
glucose DMEM, 10% FCS. For the transfection of HEK293T cells JetPei
was used (Polyplus transfections, Illkirch, France) according to
the description of the company. PARP-2 depletion and overexpression
took place as described in (Bai et al. 2007).
[0108] C2C12 cells were maintained in 4.5 g/L glucose DMEM, 10% FCS
and were differentiated in 4.5 g/L glucose DMEM, 2% horse serum for
2 days, when cells were considered myofibers. PARP-2 was depleted
from C2C12 cells using lentiviral shRNA system (MISSION Lentiviral
Vectors, Sigma-Aldrich). The vectors contained the interfering and
control sequences described in (Bai et al. 2007). C2C12 cells were
transduced with 20 MOI virus and were selected with 2.5 g/ml
puromycin. Cells withstanding puromycin selection were subcultured.
In all subsequent cell culture steps puromycin selection was
constantly maintained. The efficiency of knock-down at the mRNA
level was assessed by RT-qPCR.
[0109] mRNA and mtDNA Analysis.
[0110] Total RNA was prepared using TRIzol (Invitrogen) according
to the manufacturer's instructions. RNA was treated with DNase, and
2 .mu.g of RNA was used for reverse transcription (RT). cDNA was
purified on QIAquick PCR cleanup columns (Qiagen, Valencia, Calif.,
USA). 50.times. diluted cDNA was used for RT-quantitative PCR
(RT-qPCR) reactions (Bai et al., 2007). The RT-qPCR reactions were
performed using the Light-Cycler system (Roche Applied Science) and
a qPCR Supermix (Qiagen) with the primers for mice summarized in
Table 1 and 4, and with the primers for worms summarized on Table
7. mtDNA quantification was performed as described (Lagouge et al.,
2006) with the primers indicated in Table 2 and 5.
[0111] Immunoprecipitation, SDS-PAGE, Western Blotting.
[0112] Cells were lysed in lysis buffer (50 mM Tris, 100 mM KCl,
EDTA 1 mM, NP40 1%, nicotinamide 5 mM, Na-butyrate 1 in M, protease
inhibitors pH7.4). Proteins were separated by SDS-PAGE and
transferred onto nitrocellulose membranes. The origin of the
primary and secondary antibodies used can be found as supplemental
experimental procedures. Reactions were developed by enhanced
chemiluminescence (Amersham, Little Chalfont, UK). PGC-1.alpha.,
FOXO1 and Ndufa9 acetylation levels were analyzed by
immunoprecipitation from cellular or nuclear lysates of tissues
with anti-PGC-1.alpha. (Millipore), anti-FOXO1 (Cell Signaling,
Danvers, Mass., USA) and anti-Ndufa9 (Abcam) antibody followed by
Western blot using an acetyl-lysine antibody (Cell Signaling) that
was normalized to total PGC-1.alpha./FOXO1/Ndufa9 levels (Lagouge
et al., 2006; Rodgers et al., 2005). In HEK293T cells HA-tagged
PGC-1.alpha. was overexpressed and was immunoprecipitated using an
anti-HA. In C2C12 myotubes, FLAG-HA tagged PGC-1.alpha. was
introduced through adenoviral delivery 2 days before treatments,
then IP was performed using anti-FLAG antibody and samples were
processed as described. All blots were quantified by densitometry
using ImageJ software. Poly(ADP-Ribose) detection was performed as
previously described with slight modifications (Bai et al., PARP-1
inhibition increases mitochondrial metabolism through SIRT1
activation. Cell Metab 13, 461-468. (2011)) Briefly, PAR was
detected by using a monoclonal anti-PAR antibody (Alexis, Lausanne
Switzerland) by Western blotting of total protein lysates, using
either 50 g of total worm protein lysates, 50 g of liver protein
lysate or 200 g of muscle total protein extracts. PGC-1 acetylation
was determined using PGC-1 immunoprecipitates from 2 mg of protein
extracts, as described in Canto et al. (Canto et al., AMPK
regulates energy expenditure by modulating NAD+ metabolism and
SIRT1 activity. Nature 458, 1056 (2009)).
[0113] Chromatin Immunoprecipitation (ChIP).
[0114] ChIP was performed according to (Bai et al., 2007).
FLAG-HA-PGC-1 (Rodgers et al., 2005) was introduced by adenoviral
transfer into C2C12 myotubes after 48 h of differentiation and
cells were cultured for an additional 2 days. Cells were then
exposed to 1 M PJ34 in saline for 24 h. Thereafter ChIP was
performed using anti-FLAG (Sigma) and anti-TNF-R1 (Santa Cruz) as
described (Bai et al., 2007). Pelleted DNA was quantified by qPCR
using the primers against PDK4 and UCP-3 promoters flanking the
nuclear receptor site (Table 3 and 6). The results were normalized
for the signal of the respective inputs (vehicle/PJ34-treated) and
were expressed as a percentage. The signal of anti-TNF-R1
(non-specific antibody) was subtracted from the anti-FLAG signal
(specific) and the specific signal was plotted. Primers for ChIP
are summarized in Supplementary Table 3 and 6.
[0115] Antibodies Used for Western Blot Applications.
[0116] PARP-1 (Erdelyi et al., 2009), PAR (Alexis, Lausanne,
Switzerland), SIRT1 (Millipore), FOXO1 (Cell Signaling),
haemagglutinin (Sigma), Complex I (Ndufa9) (Abcam), Complex IV
(COXI) and V (subunit) (Molecular probes), FLAG (Sigma), and actin
(Sigma) were detected using a polyclonal rabbit antibodies. SIRT1
(Millipore), actin (Sigma), PARP-2 (rabbit polyclonal antibody
raised against full length mouse PARP-2) and H1 (kindly provided by
S. Muller, IBMC, Strasbourg). FOXO1 and SOD2 antibodies were from
Santa Cruz Biotechnology, and Acetyl-tubulin antibodies were from
Sigma Inc. Antibodies for mitochondrial markers were purchased from
Mitosciences. The secondary antibody was IgG-peroxidase conjugate
(Sigma, 1:10000).
[0117] Constructs, and Reporter Assays
[0118] SIRT1 promoter constructs were described in (Nemoto et al.,
2004), pSuper-siPARP2, pSuper-scrPARP2 and pBabe-PARP2 were
described in (Bai et al., 2007). Adenovirus for SIRT1 knockdown is
reported in (Rodgers et al., 2005).
[0119] SIRT1 Promoter Reporter Assay
[0120] HEK293T cells seeded in 6 well plates, after the depletion
or overexpression of PARP-2, were transfected with 1.6 g SIRT1
promoter reporter construct, 1 g of
pBabe/pBabe-PARP-2/pSuper-shPARP-2/pSuper-scrPARP-2 and 0.4 g
pCMV-Gal. Ten hours after transfection cells were scraped then
luciferase and -galactosidase activity were determined. Luciferase
activity was expressed as luciferase activity/-galactosidase
activity.
[0121] NAD.sup.+ NAM Determination.
[0122] NAD.sup.+ levels in cultured cells were determined using a
commercial kit (Enzychrom.TM., BioAssays Systems, CA). For tissue
samples NAD.sup.+ and NAM levels were determined as described in
(Sauve et al., 2005). In brief, to a weighed aliquot of frozen
pulverized tissue we added as standards, O.sup.18-NAD.sup.+
(typically 2.00 nmol) and O.sup.18-NAM (typically 2.00 nmol). 70 L
of ice-cold 7% perchloric acid was then added and the sample was
vortexed and sonicated three times, then centrifuged. Clear
supernatant was removed and neutralized by additions of 3 M NaOH
and 1 M phosphate buffer (pH=9), then centrifuged. Clear
supernatant was injected onto HPLC C-18 column with 20 mM ammonium
acetate eluent to separate NAD.sup.+ and NAM from other cellular
components, NAD.sup.+ and NAM peaks (260 nm absorbance) were
collected. Collections were lyophilized to dryness and subjected to
MALDI-TOF analysis. For NAD.sup.+ measurement, ratio of intensities
for m/z=664 and 666 peaks, corresponding to .sup.16O- and
.sup.18O-NAD.sup.+ isotopomers, was multiplied by 2.00 nmol and
then divided by tissue weight to determine NAD+ concentration in
the sample. For NAM the ratio of intensities for m/z=123 and 125
peaks, corresponding to .sup.16O- and .sup.18O-NAM isotopomers, was
multiplied by 2.00 nmol and then divided by tissue weight to
determine NAM concentration in the sample. Corrections were applied
for isotopic abundance. Other NAD metabolites were determined as
previously described (Yang and Sauve, Synthesis of nictotinamide
riboside and derivatives: effective agents for increasing
nictinomide adenine dinucleotide concentrations in mammalian cells.
J Med Chem 50, 6458-6461 (2006)).
[0123] Oxygen Consumption in Cultured Cells.
[0124] Cellular O.sub.2 consumption was measured using a Seahorse
bioscience XF24 analyzer with thirty biological replicates per
condition, in 24 well plates at 37.degree. C., exactly as described
(Canto et al., 2009). C2C12 were infected with an adenovirus
encoding for FLAG-HA-PGC-1 and either scramble or SIRT1 shRNAs 48 h
previous to O.sub.2 consumption measurements. Then myotubes were
treated with 1 .mu.M PJ34 for 48 h. In order to measure the
contribution of fatty acid oxidation to global O.sub.2 consumption,
total cellular O.sub.2 consumption was measured for 6 successive 2
min measurements at 5 min intervals. Then, 50 .mu.l of etomoxir (1
mM; Calbiochem, San Diego, Calif., USA) were added and, after 15
mM, 6 successive 2 min measurements were performed at 5 min
intervals. The remaining O.sub.2 consumption was attributed to
non-lipid substrates, while the difference with the initial O.sub.2
consumption values was considered due to fatty acid oxidation.
[0125] Intraperitoneal Pyruvate Tolerance Test (ipPTT)
[0126] Intraperitoneal pyruvate tolerance test was performed as
described (Yao et al., 2006). Increase in blood glucose was
expressed as percentage of baseline glucose level.
[0127] Sequence Alignment
[0128] Sequences of SIRT1 promoter was acquired for the indicated
vertebrate species from Pubmed. The initial 300 bp segment (-1 to
-300) was aligned using the ClustalW algorithm
(http://www.ebi.ac.uk/Tools/msa/clustalw2/) and the sequences
homologous to the murine -1--204 (corresponding to the two shortest
SIRT1 promoter construct) were displayed.
[0129] Cold Exposure
[0130] Cold exposure was performed as described (Lagouge et al.,
2006).
[0131] Endurance Test
[0132] Endurance exercise tests were performed as described in
Lagouge et al., 2006 on C57Bl/6J mice fed with HFD or HFD-NR for 12
weeks. Chow fed PARP-2 mice were subjected to a resistance running
test, using a variable speed belt treadmill enclosed in a plexi
glass chamber with a stimulus device consisting of a shock grid
attached to the rear of the belt (Columbus). Animals were
acclimatized to the chamber for 5 days preceding the running test.
For the habituation, mice run at 12 m/min for 10 minutes with a
5.degree. incline. For the actual test, we used a protocol at
5.degree. incline where, beginning at 10 m/min, speed increased
gradually by 1 m/min every 5 minutes. The distance run and the
number of shocks were monitored during the test, and exhaustion was
assumed when mice received more than 50 shocks in a 2.5 minutes
interval. Mice were removed from the treadmill upon exhaustion.
After high-fat diet, a few modifications were introduced:
habituation was performed for 5 days preceding the test running at
10 m/min for 10 minutes with no inclination. For the actual test,
we used a protocol with no incline where, beginning at 8 m/min,
speed increased gradually by 1 m/min every 5 minutes.
[0133] Hyperinsulinemic-euglycemic clamps were performed as
described (Lagouge et al., 2006).
[0134] Islet Size Determination
[0135] Islet size was determined on insulin-stained slides. From
each pancreas several consecutive sections (3-11) were made and all
were stained for insulin. All islets on each section were
photographed on a Zeiss Axioscope microscope and a Zeiss Axiocam
camera with special care to avoid duplicate photographing the same
islet multiple times. The area of the islets was measured using the
Image J freeware that was converted into m.sup.2 by determining the
original size of a large islet.
[0136] Mitochondrial DNA Measurement
[0137] The assay was performed as described (Lagouge et al., 2006).
DNA was extracted using the standard proteinase K digestion
following phenol-chloroform extraction. Mitochondrial and genomic
DNA was determined using specific primers in qPCR reactions
(primers summarized in Table 2 and 5.) and was expressed as
percentage of WT.
[0138] Muscle Nuclear Protein Isolation
[0139] The protocol was slightly modified, as described (Edelman et
al., 1965). Gastrocnemius muscle was removed from animals after 6
hours of fasting and was frozen immediately in liquid N.sub.2. All
manipulations were carried out on ice. Tissues were homogenized in
2 ml homogenization buffer H1 (0.32 M sucrose; 1 mM MgCl.sub.2; 0.2
mM K.sub.2HPO.sub.4; 0.6 mM KH.sub.2PO.sub.4; pH 6.8) by Ultra
TURAX (.about.1000 rpm). The homogenate was obtained in a
Potter-Elvhejem tissue grinder. Next the suspension was filtered
through a 70 .mu.m then a 40 .mu.m nylon net (BD Falcon). After
each filtration homogenate volume was adjusted to 2 ml with H1
buffer. After filtration the homogenate was centrifuged at 800 g
for 5 minutes and then the pellet was washed with additional 1 ml
H1. The pellet was desiccated at 21.degree. C. for 5 minutes and
resuspended in N2 buffer (2.15 M sucrose; 1 mM MgCl.sub.2; 3.5 mM
K.sub.2HPO.sub.4; pH 6.8). Suspension was centrifuged at 16000 g
for 2 hours and the pellet was suspended in lysis buffer (62.5 mM
TRIS pH 6.8; 2% SDS; 10% glycerol; 1 mM PMSF; 50 mM DTT; 1.times.
protease inhibitor cocktail). The pellet was resuspended in 200
.mu.l lysis buffer. The lysate was passed through a 22G needle
(20.times.) then centrifuged at 10000 rpm for 10 min. The
supernatant was used in further experiments as nuclear extract.
[0140] Determination of Protein Acetylation Status
[0141] PGC-1.alpha., FOXO1 and Ndufa9 acetylation was analyzed by
immunoprecipitation of PGC-1, FOXO1 and Nduf9a from nuclear lysates
of tissues with anti-PGC-1.alpha. (Millipore), anti-FOXO1 (1 mg,
Cell Signaling, Danvers, Mass., USA), or Ndufa9 (Abcam) antibody
followed by Western blot using an acetyl-lysine antibody (Cell
Signaling) that was normalized to total PGC-1.alpha./FOXO1/Ndufa9
(Canto et al., 2009). Tubulin acetylation was assessed by Western
blotting of total protein samples by acetylated tubulin-specific
antibody (Sigma) that was then normalized for total tubulin (Santa
Cruz).
[0142] GPR109A--Calcium Mobilization Assay.
[0143] Ready-to-Assai.TM. GRP109A Nicotinic Acid Receptor Cells
were used to measure calcium mobilization as specified by the
manufacturer (Millipore). Calcium flux was determined using
excitation at 340 and 380 nm in a fluorescence spectrophotometer
(Victor X4, Perkin Elmer) in a 180 seconds time course, adding the
ligand at 60 seconds. Internal validation was made using 0.1%
Triton X-100 for total fluorophore release and 10 mM EGTA to
chelate free calcium. Similarly, GPR109A specificity was internally
validated using control cells devoid of GRP109A overexpression.
Liver triglyceride measurement Liver triglycerides were measured
from 20 mg of liver tissue using a variation of the Folch method,
as described in Bai et al. (Bai et al., PARP-2 regulates SIRT1
expression and whole-body energy expenditure. (2011). Cell Metab
13, 450-460.
[0144] Statistics.
[0145] For the statistical analysis in the animal studies, all data
was verified for normal distribution. To assess significance we
performed Student's t-test for independent samples. Values are
expressed as mean+/-SEM unless otherwise specified. Survival
analyses were performed using the Kaplan Meier method and the
significance of differences between survival curves calculated
using the log rank test. Differences between two groups were
assessed using two-tailed t-tests. To compare the interaction
between age and genotype, two-way ANOVA tests were performed.
Calculation of mean lifespan and SEM were calculated using the R
software. Analysis of variance, assessed by Bonferroni's multiple
comparison test, was used when comparing more than two groups. The
statistical software used was GraphPad Prism 5 (GraphPad Software,
Inc.) and all p-values <0.05 were considered significant.
[0146] C. elegans Strains
[0147] C. elegans strains were cultured at 20.degree. C. on
nematode growth media agar plates seeded with E. coli strain OP50
unless stated otherwise. Strains used were wild-type Bristol N2,
RB1042 pme-1 (ok988) I, CF1038 daf-16(mu86) I, VC199 sir-2.1(ok434)
IV, KN259 (huIs33[sod-3::GFP+pRF4(rol-6(su1006))] and SJ4103
(zcIs14[myo-3::GFP(mit)]). Strains were provided by the
Caenorhabditis Genetics Center (University of Minnesota).
[0148] Worm Lifespan Analysis
[0149] Lifespan tests were performed as described in Mouchiroud et
al (L. Mouchiroud et al., Pyruvate imbalance mediates metabolic
reprogramming and mimics lifespan extension by dietary restriction
in Caenorhabditis elegans. Aging Cell 10, 39 (2011)). Briefly,
60-100 animals were used per conditions and scored every other day.
All lifespan experiments were performed at 20.degree. C. Animals
that crawled off the plate or had an <<exploded vulva>>
phenotype were censored. Treatments with PARP inhibitors--AZD2281
(also known as KU59436, olaparib), and ABT888 (also known as
veliparib)--or NAD.sup.+ precursors--nicotinamide riboside (NR) and
nicotinamide (NAM)--were added at the indicated concentration just
before pouring the plates. Animals were exposed to compounds during
the full life from eggs until death. To ensure a permanent exposure
to the compound, plates were changed twice a week. All the
compounds used in this study were dissolved in a water stock
solution.
[0150] Fluorescence Analysis
[0151] Quantification of GFP expression and endogenous gut
fluorescence were carried out according to the protocol as
described in Yamamoto et al. (Yamamoto et al., NCoR1 is a conserved
physiological modulator of muscle mass and oxidative function. Cell
147, 827 (2011)). Briefly, GFP was monitored in day 1 or day 3
adults. Fluorimetric assays were performed using a Victor X4
multilabel plate reader (Perkin-Elmer Life Science). Eighty worms
were picked at random (20 worms per well of a black-walled 96-well
plate) and each well was read four times and averaged. Each
experiment was repeated at least three times. Endogenous gut
fluorescence was monitored at day 1, day 4 and day 8 by following
the same protocol as described before.
[0152] Confocal Microscopy and Image Processing
[0153] Worms were immobilized with 6 mM solution of tetramisole
hydrochloride (Sigma, Buchs, Switzerland) in M9 and mounted on 6%
agarose pads on glass slides. Images of worms were acquired using
Zeiss LSM 700 Upright confocal microscope (Carl Zeiss AG,
Oberkochen, Germany) under non-saturating exposure conditions. All
the snapshots were taken from the same part of C. elegans: muscles
from the upper part of the worm, excluding the regions of
oesophagus and vulva. For each condition multiple worms were
observed and imaged. Image processing was performed with the Fiji
software. One of the critical issues during the imaging process was
the position of the worm, as it influences the level of background.
For the uniformity of the represented images, contrast and
brightness were adjusted in order, to eliminate the undesirable
background signal. Neither of these manipulations was affecting the
mitochondrial shape. Tracing of the mitochondrial network contour
was done by the use of Gaussian blur filter followed by the
application of Laplacian operator.
[0154] MitoSox Staining
[0155] MitoSox staining was performed as previously described with
slight modification (W. Yang, S. Hekimi, A mitochondrial superoxide
signal triggers increased longevity in Caenorhabditis elegans. PLoS
biology 8, e1000556 (2010)). Briefly, a population of 100 worms was
recovered in 1 ml of M9 buffer, washed three times to remove
residual bacteria, and resuspended in 200 .mu.l of 1:200 MitoSox
stock solution (initial stock solution was dissolved at 1 mM in
DMSO). After 20 minutes of treatment, worms were washed five times
in 1 ml of M9 buffer to eliminate the MitoSox reagent, and then
transferred in a black-walled 96-well plate for reading.
Fluorescence produced by the Mitosox reaction was measured as
described before.
[0156] Worm Respiration Assays
[0157] Oxygen consumption was measured using the Seahorse XF24
equipment (Seahorse Bioscience Inc., North Billerica, Mass.) as
described in Yamamoto et al. Typically, 200 animals per conditions
were recovered from NGM plates with M9 medium, washed three times
in 2 mLM9 to eliminate residual bacteria, and resuspended in 500
.mu.L M9 medium. Worms were transferred in 24-well standard
Seahorse plates (#100777-004) (50 worms per well) and oxygen
consumption was measured 6 times. Respiration rates were normalized
to the number of worms in each individual well. NAD measurement by
HPLC For NAD+ quantification, approximately 1000 worms were
collected in M9 buffer, washed five times to remove residual
bacteria and flash-frozen in liquid nitrogen and stored at
-80.sup.1/4C until analysis. Extraction was started by adding 250
.mu.l of 1.6M perchloric acid on frozen samples, followed by
extensive homogenization in a Qiagen tissuelyser, and
centrifugation at 20.000 g for 15 min. The supernatant was
recovered and neutralized with a 3M potassium carbonate solution.
After centrifugation, 100 .mu.l of supernatant was filtered and
used for NAD measurement using an HPLC system (Agilent) with a
Supelco LC-18-T column (Sigma), as described in Yoshino et al.
(Yoshino, K. F. Mills, M. J. Yoon, S. Imai, Nicotinamide
Mononucleotide, a Key NAD(+) Intermediate, Treats the
Pathophysiology of Diet- and Age-Induced Diabetes in Mice. Cell
Metab 14, 528 (2011)).
Example 2
PARP-1.sup.-/- Mice are Leaner and Display Increased Energy
Expenditure
[0158] A striking initial observation was that PARP-1.sup.-/- mice
on chow weighed less (FIG. 1A) and accumulated less fat than
wild-type (WT, PARP-1.sup.+/+) littermates upon aging (FIG. 1B).
This happened despite the fact that the PARP-1.sup.-/- mice
consumed significantly more food (FIG. 1C). The effects of PARP-1
deletion on body mass and food intake were observed in both males
and females (data not shown). During indirect calorimetry,
PARP-1.sup.-/- mice also consumed more O2 (FIG. 1D), suggesting
that their decreased body weight might be a consequence of
increased energy expenditure (EE). Interestingly, resting energy
expenditure (REE) was not different (FIG. 8), suggesting that the
increase could be attributed to changes at night, when the mice are
active. In line with this, spontaneous locomotor activity was
significantly increased at night in PARP-1.sup.-/- mice (FIG. 8B).
Consistently, the respiratory quotient was also higher in
PARP-1.sup.-/- mice during the dark phase (FIG. 1E), suggesting
increased glucose oxidation rates during the feeding period.
PARP-1.sup.-/- mice could also better maintain their body
temperature upon cold exposure (FIG. 1F), indicating improved
adaptive thermogenesis.
[0159] From a metabolic perspective, PARP-1.sup.-/- mice were more
glucose tolerant (FIG. 1G) and had a tendency towards lower fasting
glucose levels (4.30.+-.0.17 mM in PARP-1.sup.+/+ mice vs.
3.98.+-.0.18 mM in PARP-1.sup.-/- mice; p=0.058) despite having
similar fasting insulin levels (data not shown). When submitted to
a euglycemic-hyperinsulinemic clamp, PARP-1.sup.-/- mice did not
present any major difference in glucose infusion rates (GIR) (FIG.
1H), or hepatic glucose production (HGP) (FIG. 1I), but, supporting
their better glucose tolerance, they displayed a tendency towards
higher glucose uptake in muscle (FIG. 1J). Also In line with the
lower fat mass and improved glucose tolerance, serum triglyceride
(1.04.+-.0.07 mM in PARP-1.sup.+/+ mice vs. 0.84.+-.0.05 mM in
PARP-1.sup.-/- mice; p=0.048) and free fatty acid levels
(0.93.+-.0.09 mEq/L in PARP-1.sup.+/+ mice vs. 0.72.+-.0.03 mEq/L
in PARP-1.sup.-/- mice; p=0.040) were reduced in PARP-1.sup.-/-
mice. Overall, these results indicate that PARP-1.sup.-/- mice,
even on chow diet, have higher energy expenditure than WT mice,
resulting in a leaner phenotype and better thermogenic and
metabolic profile.
Example 3
PARP-1 Protein Levels and Activity are Regulated by Metabolic
Challenges
[0160] The striking impact of PARP-1 deletion on metabolism made us
wonder whether PARP activity would be dynamically regulated in
normal mice upon physiological changes in nutrient availability. To
test this hypothesis we analyzed whether nutrient scarcity
(fasting) or overload (high-fat diet) would have an effect on
PARP-1 activity. A 24-hr fast promoted a significant reduction in
PARP activity, as manifested by the lower PARP-1 autoPARylation
levels, which reflect global PARylation activity (Adamietz, 1987)
(FIG. 2A). This change happened in the absence of changes in total
PARP-1 levels, suggesting a lower activity of the enzyme (FIG. 2A).
In contrast, nutrient overload induced by high-fat feeding promoted
a robust increase in PARP-1 protein levels and PARP activity (FIG.
2B). Together, these data indicate that PARP-1 levels and activity
are positively regulated by nutrient availability.
Example 4
PARP-1.sup.-/- Mice are Resistant to HFD-Induced Diabesity
[0161] Given the above data, indicating that nutrient availability
dynamically regulates PARP activity, and the lean phenotype
promoted by PARP-1 deletion, it was speculated that PARP-1 could be
involved in the development of high-fat diet-induced metabolic
disease. To test this hypothesis, we fed PARP-1.sup.+/+ and
PARP-1.sup.-/- mice with a high-fat diet for two months. As
expected, PARP-1.sup.-/- mice gained less weight after high fat
feeding (FIG. 2C). The blunted body weight gain was due to a
decrease in white fat mass, as manifested by the decreased weight
of all the fat depots analyzed (FIG. 2D). The weight of other
tissues, such as pancreas, heart or kidney, was similar between the
two genotypes (FIG. 2D and data not shown). In contrast to the
moderately improved glucose tolerance of PARP-1.sup.-/- mice on
chow diet, a more pronounced reduction in the area under the curve
(AUC) in the oral glucose tolerance test was now evident in
PARP-1.sup.-/- mice upon HFD (FIG. 2E). Finally, in line with the
protection against body weight gain, the PARP-1 deletion also
rendered mice fed with HFD more insulin sensitive (FIG. 2F). As in
chow diet, serum insulin levels were not different between the two
genotypes (data not shown). Furthermore, while no differences in
serum triglyceride levels could be found after 8 weeks of HFD
between PARP-1.sup.+/+ and .sup.-/- mice (data not shown),
PARP-1.sup.-/- mice had lower serum free fatty acid levels
(0.66.+-.0.05 mEq/L in PARP-1.sup.+/+ mice vs. 0.53.+-.0.03 mEq/l
in PARP-1.sup.-/- mice: p=0.026). As a whole, these experiments
show that PARP-1.sup.-/- mice are protected against HFD-induced
obesity and metabolic disorders.
Example 5
Higher Mitochondrial Content in Brown Adipose Tissue and Muscle
from PARP-1.sup.-/- Mice
[0162] The phenotypic impact of the PARP-1 deletion on energy
expenditure and metabolic fitness suggested that the mitochondrial
activity of PARP-1.sup.-/- mice might be enhanced in metabolic
tissues, such as skeletal muscle and brown adipose tissue (BAT).
The influence of PARP-1 deletion on BAT biology was evidenced by
the fact that PARP-1.sup.-/- mice had a relatively higher amount of
BAT, when expressed as percentage of total body weight, and by the
more intense red morphological appearance of the BAT (FIG. 3A).
Furthermore, PARP-1.sup.-/- BAT contained more mitochondria based
on the increased mitochondrial DNA content (FIG. 3B). This higher
mitochondrial DNA content in PARP-1.sup.-/- mice was in line with
increased expression of genes involved in mitochondrial uncoupling
(Uncoupling protein-1 (UCP1) and UCP3), fatty acid oxidation
(Medium chain acylcoenzyme A dehydrogenase, (MCAD)) and respiration
(Ndufa2, Ndufb2, Ndufb5, Cytochrome c (cyt c), COX17) (FIG. 3C).
Additionally, the expression of Deiodinase-2 (Dio2) was higher in
PARP-1.sup.-/- mice, suggesting increased thyroid hormone
activation in the BAT (FIG. 3C). Furthermore, the increased
mitochondrial content in PARP-1.sup.-/- BAT was also evidenced by
transmission electron microscopy (FIG. 3D) and by the higher
protein expression of specific subunits from different respiratory
complexes (FIG. 3E).\
[0163] As in BAT, the protein levels of mitochondrial respiratory
complexes were also markedly induced in the gastrocnemius muscle of
PARP-1.sup.-/- mice (FIG. 3F). In line with this, succinate
dehydrogenase (SDH) staining (FIG. 3G) and expression analysis of
muscle fiber isotype genes (Troponin I (Trop I), Myosin heavy chain
I (MHCI); FIG. 3H) indicated an increase in the number of oxidative
fibers, characterized by a higher mitochondrial content than
glycolytic fibers. Similar to what was observed in BAT, the
increased mitochondrial content was linked to an upregulation of
the expression of genes encoding mitochondrial proteins (FIG.
3H).
[0164] Another crucial tissue for whole body metabolism is the
liver and we investigated the expression of a large set of genes
encoding for transcription factors (Estrogen receptor
related--(ERR), Peroxisome proliferator activated receptor--(PPAR
.alpha.), Peroxisome-proliferator-activated receptor-gamma
co-activator 1 (PGC-1.alpha.), Sterol regulatory element-binding
protein (SREBP1)), proteins involved in mitochondrial respiration
(Ndufa2, Ndufa3, cyt c, ATP5g1), in fatty acid oxidation (MCAD,
ACO), in fatty acid synthesis (Malic enzyme, acetyl-CoA
carboxylase-1 and -2 (ACC-1, -2)) and in glucose metabolism
(Phosphoenol pyruvate carboxykinase (PEPCK), Glucokinase (GK),
glucose-6 phosphatase (G6Pase)) (FIG. 9A). The lack of significant
changes in the expression of this metabolic geneset suggested that
the absence of PARP-1 has only a minor metabolic impact in the
liver, potentially explained by the very low expression of PARP-1
in the liver relative to skeletal muscle or BAT (FIG. 9B).
Example 6
BAT Mice and Muscle from PARP-1.sup.-/- Display Higher NAD.sup.+
Content and SIRT1 Activity
[0165] The above results illustrate that PARP-1.sup.-/- mice have
more mitochondria in BAT and in muscle, a tissue which was also
enriched in oxidative fibers. This phenotype resembles many of the
features expected from SIRT1 activation (Baur et al., 2006; Feige
et al., 2008; Lagouge et al., 2006; Milne et al., 2007). PARP-1 is
a major NAD.sup.+-consumer in the cell (Shieh et al., 1998; Sims et
al., 1981), making it tempting to speculate that in limits
NAD.sup.+ normal conditions, PARP-1 availability for SIRT1.
[0166] Therefore, the lack of PARP-1 activity could lead to higher
NAD.sup.+ levels and, consequently, activate SIRT1. PARP-1 is
considered to be the enzyme that drives most of the PARP activity
in the cell (Shieh et al., 1998; Sims et al., 1981). In agreement
with this, the ablation of PARP-1 reduced PARylation in both BAT
and muscle (FIG. 4A). The expression of the other known PARP
enzymes was not induced in BAT and muscle of PARP-1.sup.-/- mice
(FIGS. 3A-B), explaining the lack of compensation on global
PARylation. Importantly, terminal dUTP nick-end labeling (TUNEL)
assays revealed that the number of DNA strand breaks was not
increased in PARP-1.sup.-/- tissues (data not shown), confirming
previous observations indicating that PARP-1 deletion by itself
does not lead to the accumulation of DNA damage (Allinson et al.,
2003; Fong et al., 2009). Consequent to the attenuated
NAD.sup.+-consuming PARylation activity, a robust increase in NAD+
content was observed in the BAT and muscle of PARP-1.sup.-/- mice
(FIG. 4B). Interestingly, the levels of nicotinamide (NAM), a
NAD.sup.+-derived metabolite that inhibits sirtuin activity
(Bitterman et al., 2002), were not altered (FIG. 4C), indicating
that the effect is specific for NAD+. We next tested the increase
in NAD+ whether levels affected the activity of SIRT1.
[0167] Indicative of SIRT1 activation and supporting the increase
in mitochondrial content, PGC-1 acetylation levels in BAT (FIG. 4D)
and skeletal muscle (FIG. 4E) of PARP-1.sup.-/- mice were
significantly reduced by .about.40% and .about.90%, respectively,
compared to PARP-1+/+ mice. The deacetylation of another SIRT1
target, forkhead box O1 (FOXO1), was also significantly reduced by
.about.60% in BAT (FIG. 4D) and .about.40% in muscle (FIG. 4E),
further supporting that the loss of PARP-1 activity leads to
generalized SIRT1 activation. Remarkably, SIRT1 protein levels were
robustly increased in muscle and BAT from PARP-1.sup.-/- mice
(FIGS. 4D and 4E) contributing to elevated SIRT1 activity. While
not significantly different in BAT (FIG. 4D), PGC-1 protein levels
were also higher in the muscles from PARP-1.sup.-/- mice (FIG.
4E).
[0168] It is important to note that altered NAD.sup.+ levels could
potentially impact on the activity of not just SIRT1, but also
other sirtuins. Hence, we explored the activity of the closest
SIRT1 homologs that are also expressed in different cellular
compartments: SIRT2 (North et al., 2003) and SIRT3 (Schwer et al.,
2002), which act as cytoplasmatic and mitochondrial sirtuins,
respectively. The acetylation level of tubulin, an established
SIRT2 target (North et al., 2003), was not altered in the
gastrocnemius muscle of PARP-1-/- mice (FIG. 4 F). Likewise, the
acetylation levels of Complex I, a target for SIRT3 (Ahn et al.,
2008) even showed a slight, but not significant, tendency to
increase on the PARP-1.sup.-/- muscles (FIG. 4G). These
observations indicate that not all sirtuins increase their activity
in response to the changes in NAD.sup.+ levels induced by PARP-1
ablation.
Example 7
Knocking-Down PARP-1 in Cultured Cells Enhances Oxidative
Metabolism
[0169] Given the effects of the somatic ablation of PARP-1 on SIRT1
activity and mitochondrial content in transgenic mice, we next
evaluated whether reducing PARP-1 activity in an acute fashion
could constitute a useful mechanism to increase cellular NAD.sup.+
levels and improve energy metabolism. For this purpose, we
knocked-down PARP-1 expression in HEK293T cells. With this
approach, we reduced PARP-1 protein levels by .about.80%, which
dramatically reduced global intracellular PARP activity as
manifested in the low auto-poly(ADP-ribosyl)ation of PARP-1 (FIG.
5A). The reduction of PARP activity in this cell model perfectly
recapitulated all our in vivo findings. First of all, the reduction
in PARP-1 activity increased the NAD.sup.+ content (FIG. 5B) and
subsequently enhanced SIRT1 function, as evidenced by the strong
decrease in PGC-1 acetylation levels (FIG. 5C). Importantly, this
change happened in the absence of changes in SIRT1 protein levels
(FIG. 5C), indicating that the changes NAD.sup.+ might act as the
main drivers of SIRT1 activity. These changes in SIRT1 and PGC-1
activity culminated in robust increase in mitochondrial DNA content
(FIG. 5D), mitochondrial-related gene expression (CPT-1b, MCAD,
UCP3 and PPAR) (FIG. 5E), and cellular O.sub.2 consumption (FIG.
5F). These observations indicate that reduction of PARP-1 levels,
even in an acute fashion, activate SIRT1 and promote mitochondrial
biogenesis and oxidative metabolism. Importantly, the majority of
this oxidative phenotype was dependent on SIRT1 action, as
evidenced by results showing that most of the metabolic effects
elicited by PARP-1 depletion were lost when SIRT1 was
simultaneously knocked-down (FIG. 5D-F).
[0170] We next aimed to consolidate these observations with the use
of MEF cells from PARP-1.sup.+/+ and PARP-1.sup.-/- mice. In line
with the data obtained from the PARP-1 knockdown experiments, MEFs
from PARP-1.sup.-/- mice showed enhanced O.sub.2 consumption (FIG.
11A), increased mitochondrial content (FIG. 11B), mitochondrial
membrane potential (FIG. 11C) and higher expression of genes
involved in mitochondrial function (e.g. PGC-1, NDUF5b, cyt c,
COX17, UCP-2, mCPT-1, ACO) (FIG. 11D). In line with the
observations made on tissues from PARP-1 null mice, PARP-1.sup.-/-
MEFs displayed increased SIRT1 protein content (FIG. 11E).
Together, the observations in two different cellular models fully
support that a reduction in PARP-1 levels promotes mitochondrial
gene expression and oxidative metabolism.
Example 8
Pharmacological Inhibition of PARP Activity Enhances Oxidative
Metabolism Via SIRT1
[0171] To test the dynamic interplay between SIRT1 and PARP-1, we
exposed C2C12 myotubes to H.sub.2O.sub.2 (500 .mu.M, 6 hrs).
H.sub.2O.sub.2 is a very well-known activator of PARP-1
(Schraufstatter et al., 1986) and, accordingly, increased protein
PARylation, as manifested by the slow migration band, in the
absence of changes in total PARP-1 levels (FIG. 6A). This increase
in PARP-1 activity led to a marked decrease in intracellular
NAD.sup.+ levels (FIG. 6B). It is important to note that
H.sub.2O.sub.2 treatment did not affect SIRT1 protein levels (FIG.
6B). However, due to the lower NAD.sup.+ bioavailability, SIRT1
activity was markedly lower, as testified by the hyperacetylation
of PGC-1a (FIG. 6C). We also tested whether inhibition of PARP
activity, using the global PARP activity inhibitor PJ34 (Garcia et
al., 2001), would prevent the decrease SIRT1 activity during
H.sub.2O.sub.2 treatment (FIG. 6A). Confirming our hypothesis, PARP
inhibition prevented the decrease in intracellular NAD.sup.+ (FIG.
6B) and enhanced SIRT1 function (FIG. 6C), despite the fact that
SIRT1 protein levels did not change.
[0172] While the above results clearly indicate that PARP-1
activation limits SIRT1 activity and that PARP inhibitors relieve
this limitation, it is generally assumed that basal PARP activity
is rather low. However, recent evidence indicates that PARP-1
activity is not necessarily linked to DNA-damage and that it even
fluctuates in a circadian fashion (Asher et al., 2010). Therefore
it is tempting to speculate that prolonged PARP inhibition, even in
the absence of DNA damage will favor NAD.sup.+, accumulation and,
potentially, SIRT1 activity. In line with this hypothesis,
inhibition of PARP activity with PJ34 led to a gradual increase in
NAD.sup.+, becoming significant 24 hrs after the initiation of the
treatment (FIG. 6D). After 24 hrs, PJ34 treatment robustly
decreased basal PARP activity (FIG. 6E), while PARP-1 protein and
mRNA levels remained unchanged (FIG. 6E and data not shown). The
increase in NAD promoted by PJ34 at 24 hrs happened in a dose
dependent manner (FIG. 6F). We further confirmed the increase in
NAD.sup.+ content upon PARP-1 inhibition using another,
structurally unrelated PARP inhibitor, TIQ-A (data not shown)
(Chiarugi et al., 2003), this dose-dependent increase in NAD.sup.+
importantly correlated with SIRT1 activation, as illustrated by the
deacetylation of PGC-1.alpha. (FIG. 6F). It is important to note
that, while compounds like resveratrol impact on SIRT1 through
AMP-activated protein kinase (AMPK) (Canto et al., 2010; Urn et
al., 2010), PJ34 does not alter AMPK activity as reflected by the
unchanged acetyl-CoA carboxylase (ACC) phosphorylation levels (FIG.
6G). Similarly, 24 hrs treatment with PJ34 did not change SIRT1
protein levels (FIG. 6G). Together, these observations suggest that
it is mainly the increase in NAD.sup.+ promoted by PJ34 that is
responsible for the activation of SIRT1. Consequent to PGC-1.alpha.
deacetylation and activation, PJ34 treatment increased PGC-1.alpha.
recruitment to target-genes, as manifested in ChIP experiments
using the PDK4 promoter (FIG. 12A). This led to the induction of
mitochondrial biogenesis, as manifested in the increased expression
of several mitochondrial genes, including Ndufa2, Ndufa3, MCAD,
PDK4 and UCP3 (FIG. 6H), and higher mitochondrial DNA content (FIG.
12B). In addition, PJ34-treated myotubes displayed enhanced
oxidative metabolism, as testified by the higher mitochondrial
potential (FIG. 12C) and O.sub.2 consumption rates (FIG. 6 I). A
closer analysis indicated that the increase in O.sub.2 consumption
could not be attributed solely to an increase in fatty acid
oxidation, but rather to the combination of enhanced oxidation of
lipid and non-lipid substrates (FIG. 12D).
[0173] Interestingly, when SIRT1 expression was knocked-down in
C2C12 myotubes by the use of specific shRNAs the effects of PJ34 on
PGC-1.alpha. acetylation, were blunted (FIG. 6G). The major role of
SIRT1 in mediating PJ34-induced PGC-1.alpha. deacetylation was
further MEFs from SIRT1-/- confirmed in mice, where PJ34 was
totally unable to decrease PGC-1.alpha. acetylation levels (FIG.
12E). In line with the impaired PGC-1.alpha. activation,
mitochondrial gene expression and O.sub.2 consumption, were also
largely blunted by the SIRT1 knock-down in C2C12 myotubes (FIG.
6H-I) and in SIRT1 MEF cells (FIG. 12F-G), indicating that SIRT1 is
an important mediator of PJ34 actions. It must be noted, however,
that PJ34-treatment also has SIRT1-independent effects, as
reflected by the persistent increase in UCP3 mRNA expression even
when SIRT1 expression was knocked-down (FIG. 6H). This could be
explained by the fact that PJ34-induced increase in UCP3 expression
is not regulated through the binding of PGC-1 to its promoter, as
evidenced by the ChIP experiment (FIG. 12A). These results indicate
that the acute pharmacological inhibition of PARP activity in
cellular models recapitulates the phenotypic characteristics of the
PARP-1.sup.-/- mice, and underscores that most of these effects are
mediated by SIRT1. The metabolic effects of PJ34 in C2C12 cells
encouraged us to test whether these effects also persisted in vivo.
To explore this possibility, we injected mice with PJ34 (10 mg/kg)
twice a day for 5 days. The treatment did not cause a difference in
body weight or in food consumption (data not shown), but was enough
to robustly decrease PARP activity in BAT and muscle (FIGS. 7A and
B, respectively). Importantly, neither SIRT1 protein levels nor
AMPK activity, as reflected by the phosphorylation levels of ACC,
were affected by PJ34 treatment (FIGS. 7A and B). Consequent to the
decrease in PARPactivity, NAD+ levels edged up in PJ34-treated
animals, even though the increase was only statistically
significant in muscle (FIG. 7C-D). This increase in NAD.sup.+
levels correlated with higher SIRT1 activity, as manifested by the
reduced PGC-1.alpha. acetylation levels (FIG. 7C-D). While both BAT
(FIG. 7E) and muscle (FIG. 7F) displayed a tendency to a higher
expression of a number of mitochondrial genes (such as Ndufa2,
Ndufa5, UCP2 and UCP3), the increase was more robust in muscle than
in BAT, in correlation with the higher increase in NAD.sup.+. In
muscle, the increase in mitochondrial gene expression induced by
PJ34 was accompanied by an increase in Myoglobin mRNA levels, which
facilitates oxygen delivery into muscle fibers (FIG. 7F). In BAT,
and in line with the results observed in PARP-1.sup.-/- mice, Dio2
mRNA was significantly induced by PJ34, suggesting thyroid hormone
activation (FIG. 7E). Interestingly, and despite the short duration
of the treatment, PJ34 caused a tendency to improve the serum
metabolite profile, as manifested by the reduction in blood glucose
(9.94.+-.0.28 mM in vehicle vs. 9.19.+-.0.58 mM with PJ34; p=0.05),
triglycerides (1.21.+-.0.08 mM in vehicle vs. 1.11.+-.0.04 mM with
PJ34; p=0.08) and free fatty acid levels (1.59.+-.0.06 mEq/L in
vehicle vs. 1.44.+-.0.03 mEq/L with PJ34; p=0.03). All these data
indicate that PJ34 treatment also in vivo phenocopies part of the
oxidative features induced by PARP-1 gene deletion.
Example 9
Role of PARP-2 as a Regulator of SIRT1 Activity
[0174] In order to examine the potential role of PARP-2 as a
regulator of SIRT1 activity, we generated C2C12 myocytes stably
transfected with either a scrambled or a PARP-2 shRNA. PARP-2 mRNA
and protein content is reduced by 80% in myotubes from cells
carrying the PARP-2 shRNA (FIG. 13A). We next evaluated whether
this deficiency in PARP-2 activity affects NAD.sup.+ homeostasis.
While inhibition of total PARP activity with the inhibitor
NAD.sup.+ PJ34 leads to increased intracellular content, a
reduction in PARP-2 by itself did not affect (FIG. 19) NAD.sup.+
total (FIG. 13B) or mitochondrial levels.
[0175] Similarly, knocking down PARP-2 did not prevent
H.sub.2O.sub.2-induced NAD.sup.+ depletion, while global inhibition
of PARP activity with PJ34 did (FIG. 13B). To further sustain our
observations we analyzed the impact of the PARP-2 knock-down on
global PARP activity by checking H2O2-induced protein PARylation.
While PJ34 completely reversed H.sub.2O.sub.2-induced PARylation,
the knock-down of PARP-2 could not prevent protein hyperPARylation
(FIG. 13C). These results confirm that PARP-2 is a secondary PARP
activity in the cell, as already demonstrated previously (Ame et
al., 1999; Shieh et al., 1998). Furthermore, it also suggests that
PARP-2 depletion has little impact on NAD.sup.+ homeostasis.
[0176] Given the absence of an impact on NAD.sup.+ homeostasis, it
was surprising to observe that myotubes in which PARP-2 had been
knocked down, displayed higher SIRT1 activity, as demonstrated by
reduced PGC-1 acetylation (FIG. 13D, top panels). We could not find
any direct interaction between PARP-2 and SIRT1 (FIG. 19 B),
indicating that changes in SIRT1 activity are not likely to happen
through direct post-translational modification by PARP-2. Rather,
the increase in SIRT1 activity was linked to increased SIRT1
content (FIG. 1D, bottom panels). The increase in SIRT1 protein was
concomitant to an increase in SIRT1 mRNA levels (FIG. 13E). To
explore why SIRT1 mRNA levels were increased by transcriptional
induction, we used a reporter construct in which serial deletions
of the mouse SIRT1 promoter region controlled luciferase expression
(FIG. 13F). These studies demonstrated that knocking down PARP-2
promoted a 2-fold increase in SIRT1 transcription through the very
proximal promoter region (-91 bp), an effect that was maintained
for the whole upstream regulatory region that was analyzed (FIG.
13F). In chromatin immunoprecipitation (ChIP) assays, PARP-2 was
shown to bind directly to the proximal SIRT1 promoter (region
comprised between the transcription start site and -91 bp) in C2C12
myotubes (FIG. 13G). The direct binding of PARP-2 on the SIRT1
promoter was also observed in a non-murine cell line, like 293HEK
cells (FIG. 19C), as this proximal -91 bp region is extremely
conserved along evolution (FIG. 19D). All these results suggest
that PARP-2 acts as a direct negative regulator of the SIRT1
promoter. Consequently, a reduction of PARP-2 levels induces SIRT1
transcription, leading to higher SIRT1 protein levels and activity.
An expected consequence of this increase in SIRT1 activity is that
a reduction in PARP-2 content should lead to higher mitochondrial
gene expression, by the activation of PGC-1.alpha. through
deacetylation, and to increased O.sub.2 consumption. This
hypothesis turned out to be correct, as cellular O.sub.2
consumption was increased in PARP-2 knock-down cells (FIG. 13H),
concomitant to the increase in expression of genes related to lipid
and mitochondrial metabolism, such as Medium Chain Acyl coenzyme A
Dehydrogenase (MCAD), NADH Dehydrogenase [Ubiquinone] 1 alpha
subcomplex subunit 2 (Ndufa2) and Cytochrome C (Cyt) (FIG. 13I).
Furthermore, using adenoviruses encoding for a shRNA for SIRT1, we
demonstrated that the increase in SIRT1 activity contributed in a
major fashion to the oxidative phenotype of PARP-2 deficient
myotubes (FIG. 13H-I).
Example 10
Knocking-Down PARP-2 in Mice Promotes an Increase in the Use of
Fat
[0177] All the experiments above illustrate that reducing PARP-2
activity might be useful to increase SIRT1 activity and,
consequently, potentiate oxidative metabolism. In order to gain
further insight in this mechanism, we next examined the metabolic
profile of the PARP-2.sup.-/- mice. PARP-2.sup.-/- mice were
smaller and leaner then their PARP-2.sup.+/+ littermates (FIG.
14A). The fact that there was no difference in food intake between
the PARP-2.sup.-/- and PARP-2.sup.+/+ mice (FIG. 14B) and that
spontaneous locomotor activity was lower in the PARP-2.sup.-/- mice
(FIG. 214C), suggested that the difference in weight gain was due
to altered energy expenditure (EE). Indirect calorimetry, however,
only indicated a slight tendency towards a higher O.sub.2
consumption in chow fed PARP-2.sup.-/- mice compared to wild type
littermates under basal conditions (FIG. 14D). Interestingly, RQ
values indicate that during the dark phase PARP-2.sup.-/- mice use
lipid substrates as energy source at proportionally higher rates
than the PARP-2.sup.+/+ littermates (FIG. 14E). Strikingly,
PARP-2.sup.-/- mice were mildly hyperglycemic in both fed and
fasted states (FIG. 2F), linked to a tendency towards lower blood
insulin levels in both fed (2.52.+-.0.24 .mu.g/L for PARP-2.sup.+/+
vs. 1.77.+-.0.13 .mu.g/L for PARP-2.sup.-/- mice) and fasted
(0.77.+-.0.07 .mu.g/L for PARP-2.sup.+/+ vs. 0.71.+-.0.11 .mu.g/L
for PARP-2.sup.-/- mice) states. Overall, these results illustrate
that PARP-2 deletion promotes an increase in the use of fat as main
energy source, associated with a leaner phenotype.
Example 11
SIRT1 mRNA and Protein Levels were Increased in Muscles from
PARP-2.sup.-/- Mice
[0178] At the molecular level, PARP-2 deletion was not linked to
higher DNA damage in either young or old mice (FIG. 20A). In line
with these in vitro data, we could not detect a significant change
in protein PARylation in PARP-2.sup.-/- mice, as determined by
western blotting (FIG. 15A). In contrast to the data from C2C12
myotubes, PARP-2.sup.-/- muscles contained more NAD.sup.+ (FIG.
15B). The data from cultured myotubes suggests that the increase in
NAD.sup.+ levels observed in muscle tissue might be secondary to
the leaner phenotype rather than a direct consequence of the
reduction in PARP-2 function per se. In line with the role of
PARP-2 as a negative regulator of the SIRT1 promoter, SIRT1 mRNA
and protein levels were increased in muscles from PARP-2.sup.-/-
mice (FIG. 15C). The combination of higher NAD.sup.+ and higher
SIRT1 protein provides an excellent scenario to increase SIRT1
activity. Confirming this hypothesis, the acetylation levels of two
different SIRT1 substrates, the peroxisome proliferator-activated
receptor (PPAR) coactivator-1 (PGC-1) (FIG. 15D) and the forkhead
box O1 (FOXO1) transcription factor (FIG. 15E), were markedly
decreased in muscles from PARP-2.sup.-/- mice. Importantly, the
acetylation status of SIRT2 and SIRT3 targets, such as tubulin and
Ndufa9, respectively, was not affected by PARP-2 deletion,
indicating that PARP-2.sup.-/- deletion is not affecting the
activity of the closest SIRT1 homologs (FIG. 20B).
Example 12
PARP-2 Deletion Influences Mitochondrial Biogenesis
[0179] PGC-1.alpha. and FOXO1 are transcriptional activators
strongly linked to the regulation of mitochondrial biogenesis and
oxidative metabolism. Consequent to their activation through
deacetylation, the expression of transcriptional regulators of
oxidative metabolism (PGC-1.alpha.), of biomarkers of oxidative
muscle fibers (troponin I (tpnI)), and of mitochondrial proteins
(succinate dehydrogenase (SDH), uncoupling protein 2 (UCP2)) and
lipid oxidation enzymes (malonyl-CoA decarboxylase (MCD), MCAD)
were increased in gastrocnemius muscle of the PARP-2.sup.-/- mice
(FIG. 15F). The increase in mitochondrial content was further
evidenced by the higher mitochondrial DNA content (FIG. 15G) and by
the more prominent mitochondria observed upon transmission electron
microscopy analysis of the gastrocnemius muscle (FIG. 15H). The
increased mitochondrial biogenesis clearly promoted a more
oxidative phenotype of the PARP-2.sup.-/- muscles, as reflected by
the prominent increase in SDH positive oxidative muscle fibers
(FIG. 15I). As a physiological consequence of this increased
oxidative muscle profile, PARP-2.sup.-/- mice performed much better
than their PARP-2.sup.+/+ littermates on a treadmill endurance test
(FIG. 15J). As a whole, these results indicate that PARP-2 deletion
promotes mitochondrial biogenesis in muscle, increasing the
oxidative and endurance profile of the fibers.
[0180] We also explored whether PARP-2 deletion could also
influence mitochondrial biogenesis in other highly metabolic
tissues, such as in brown adipose tissue (BAT) and liver. In BAT,
despite higher SIRT1 content (FIG. 3A), we were unable to detect
changes in the expression of the main metabolic genes (FIG. 21B).
Supporting the minor impact of PARP-2 deletion on BAT function,
body temperature dropped similarly in PARP-2.sup.+/+ and
PARP-2.sup.-/- mice upon cold exposure (FIG. 21C). This suggested
that BAT is unlikely to contribute significantly to the differences
in EE observed in the PARP-2.sup.-/- mice. In contrast, PARP-2
deletion had strong effects on the expression of diverse regulators
of mitochondrial metabolism in the liver, including PGC-1.alpha.,
PGC-1, FOXO1, PPAR, estrogen-related receptor (ERR) and Cytochrome
C oxidase subunit II (COXII) (FIG. 16A). Consistently,
PARP-2.sup.-/- livers displayed a higher mitochondrial content, as
evidenced by the increase in mitochondrial DNA levels (FIG. 16B)
and by the appearance of more mitochondria upon electron microscopy
(FIG. 16C). As in muscle, liver NAD.sup.+ content was higher in
PARP-2.sup.-/- mice (FIG. 16D), which, together with the higher
amounts of SIRT1 protein, translated into increased SIRT1
activation (FIG. 16E). In line with what was observed in muscle, no
changes in the activity of SIRT2 and SIRT3, the closest SIRT1
homologs, was detected (FIG. 22A). The observation that
PARP-2.sup.-/- livers had a tendency towards a reduced triglyceride
content both upon oil red O staining (FIG. 22B) and direct
biochemical measurement (FIG. 16F) is consistent with the induction
of oxidative metabolism. Despite the increase in
phosphoenolpyruvate carboxykinase (PEPCK) expression in
PARP-2.sup.-/- mice and the increased capacity of liver for
oxidative metabolism, PARP-2.sup.-/- mice responded similar to
PARP-2.sup.+/+ littermates upon a pyruvate-tolerance test (FIG.
22C), probably due to the similar expression of another
rate-limiting enzyme, the glucose-6-phosphatase (G6Pase) (FIG.
16A).
Example 13
PARP-2.sup.-/- Mice are Protected from Body Weight Gain and Insulin
Resistance Upon High-Fat Feeding
[0181] The increased mitochondrial biogenesis and oxidative
phenotype observed in the skeletal muscle and liver of
PARP-2.sup.-/- mice incited us to test how these mice would respond
to high-fat diet (HFD) feeding. PARP-2.sup.-/- mice were protected
against weight gain when fed a HFD (FIG. 17A), despite a similar
food intake (FIG. 17B). This leaner phenotype was associated with a
reduced body fat mass, as evidenced by Echo-MRI analysis (FIG.
17C). This reduction in fat content was clearly more pronounced
(20% decrease) in the epidydimal fat depots, which is equivalent to
visceral fat in man, than in the subcutaneous fat pads (FIG. 17D).
The weight of the PARP-2.sup.-/- livers was also markedly reduced
(FIG. 17D), consequent to a lower triglyceride accumulation (FIG.
23A-B). Accentuating what was observed in chow-fed mice,
PARP-2.sup.-/- mice on high fat diet displayed now significantly
higher O.sub.2 consumption rates (FIG. 17E). The increase in
VO.sub.2 was not due to increased activity (FIG. 17F), indicating
that high-fat fed PARP-2.sup.-/- mice have higher basal EE. As
expected, the expression of the transcriptional regulators
governing EE (SIRT1, PGC-1), was increased in gastrocnemius from
PARP-2.sup.-/- mice when compare d to their PARP-2.sup.+/+
littermates after the HFD (FIG. 17G). The expression of several
genes involved in fatty acid uptake and oxidation (muscle carnitine
palmitoyltansferase 1b (mCPT1b), peroxisomal acyl-coenzyme A
oxidase 1 (ACOX1), MCD, MCAD), mitochondrial electron transport and
oxidative phosphorylation (Ndufa2, Cyt C, COXIV) followed a similar
pattern as these transcriptional regulators and were maintained at
a higher level in the PARP-2.sup.-/- muscle (FIG. 17G). Consequent
to the much leaner and oxidative phenotype, PARP-2.sup.-/- mice
remained more insulin-sensitive than their wild-type littermates
after high-fat feeding (FIG. 17H), and their endurance performance
was markedly better (data not shown). These results clearly
indicate that PARP-2.sup.-/- mice are protected from body weight
gain and insulin resistance upon high-fat feeding, linked to a
better muscle oxidative phenotype.
Example 14
PARP-2.sup.-/- Were More Glucose Intolerant Compared to Their
PARP-2.sup.+/+ Littermates after High-Fat Feeding
[0182] To our surprise, despite their lower body weight and higher
insulin sensitivity, PARP-2-/- mice were more glucose intolerant
compared to their PARP-2.sup.+/+ littermates after high-fat feeding
(FIG. 18A), and still displayed fasting hyperglycemia
(172.44.+-.20.11 mg/dL for PARP-2.sup.+/+ vs. 203.34.+-.10.26 mg/dL
for PARP-2.sup.-/-). The fact that PARP-2.sup.-/- mice are also
more insulin sensitive (FIG. 17H) suggested that this glucose
intolerance could be related to defects in the insulin-release upon
a glucose load. Confirming this hypothesis, the insulin peak after
an intraperitoneal glucose injection in PARP-2.sup.-/- mice was
blunted in PARP-2.sup.-/- mice (FIG. 18B). Furthermore, fasting
blood insulin levels were lower in PARP-2.sup.-/- mice
(0.87.+-.0.24 .mu.g/L for PARP-2.sup.+/+ vs. 0.58.+-.0.16 .mu.g/L
for PARP-2.sup.-/- mice). These observations led us to examine the
pancreas from PARP-2.sup.-/- mice. High-fat diet increased the
pancreatic mass in wild-type mice, but not in PARP-2 deficient mice
(FIG. 18C). Histological analysis of the pancreas of PARP-2.sup.+/+
and PARP-2.sup.-/- mice revealed that islet size was smaller in
PARP-2.sup.-/- mice after high-fat feeding (FIGS. 18D and 18E).
This reduction in islet size translated into a robust reduction in
pancreatic insulin content (FIG. 18F). When pancreatic gene
expression was analyzed in pancreas from PARP-2.sup.+/+ and
PARP-2.sup.-/- mice, it became evident that, in addition to an
increase in some mitochondrial-related genes (mitochondrial
transcription factor A (TFAm), citrate synthase (CS)), the pancreas
of PARP-2.sup.-/- mice had severe reductions in the expression of a
number of key genes for pancreatic function (such as glucokinase
(GK) and Kir6.2) and proper-cell growth (pancreatic and duodenal
homeobox 1 (pdx1)) (FIG. 18G). Given the reduced insulin content
and pdx1 expression it was also not surprising that expression of
the insulin gene (Ins) was decreased in the PARP-2.sup.-/- pancreas
(FIG. 18G). As in other tissues, PARP-2 deletion led to higher
SIRT1 protein levels in pancreas (FIG. 18H), which translated not
only into higher mitochondrial protein content, as manifested by
complex I (39 kDa sbunit) and complex III (47 kDa subunit) levels,
(FIG. 18H) but also in the constitutive deacetylation of FOXO1
(FIG. 18H). NAD.sup.+ levels were similar in pancreas form
PARP-2.sup.+/+ and .sup.-/- mice (FIG. 24A) The deacetylation and
activation of FOXO1 could underpin the pancreatic phenotype of the
PARP-2 mice, as FOXO1 activity compromises pancreatic growth by
acting as a negative regulator of pdx1 (Kitamura et al., 2002).
Altogether, these results illustrate that high-fat diet leads to
altered expression of key genes involved in pancreatic-cell
proliferation and function in PARP-2 deficient mice, culminating
into a reduced pancreatic islet size and insulin content,
explaining the glucose intolerance despite their leaner and more
insulin-sensitive phenotype.
Example 15
NR Increases Intracellular and Mitochondrial NAD.sup.+ Content in
Mammalian Cells and Tissues
[0183] NR treatment dose-dependently increased intracellular
NAD.sup.+ levels in murine and human cell lines (FIG. 26A), with
maximal effects at concentrations between 0.5 and 1 mM. In C2C12
myotubes, the K.sub.m for NR uptake was 172.3.+-.17.6 .mu.M, with a
V.sub.max of 204.2.+-.20.5 pmol/mg of protein/min. Unlike NA, both
NR and another well-described NAD.sup.+ precursor, NMN (Revollo et
al., 2007), did not activate GPR109A (FIG. 26B), hence constituting
valuable candidates to increase NAD.sup.+ levels without activating
GPR109A. Strikingly, the ability of NR to increase intracellular
NAD.sup.+ in mammalian cells was, at least, similar to that of
these other precursors (FIG. 26C). We next evaluated the efficacy
of NR, NMN and NA to increase NAD.sup.+ in vivo by supplementing
mouse chow with NR, NMN or NA at 400 mg/kg/day for one week. All
compounds increased NAD.sup.+ levels in liver, but only NR and NA
significantly enhanced muscle NAD.sup.+ content. (FIG. 26D). These
results illustrate how NR administration is a valid tool to boost
NAD.sup.+ levels in mammalian cells and tissues without activating
GPR109A.
[0184] Given the existence of different cellular NAD.sup.+ pools
and the relevance of mitochondrial NAD.sup.+ content for
mitochondrial and cellular function, we also analyzed whether NR
treatment would affect mitochondrial NAD.sup.+ levels. In contrast
to what has been observed with other strategies aimed to increase
NAD.sup.+ bioavailability, such as PARP inhibition, we found that
mitochondrial NAD.sup.+ levels were enhanced in cultured cells
(FIG. 26E) and mouse liver (FIG. 26F) after NR supplementation.
This is, to our knowledge, the first nutritional intervention that
increases mitochondrial NAD.sup.+ levels.
[0185] To further solidify our data, we also wondered whether the
enhanced NAD.sup.+ levels upon NR treatment could derive from
alterations in the NAD.sup.+ salvage pathway or PARP activity.
However, we could not see any change in Nampt mRNA or protein
content in response to NR treatment (FIG. 26G). Similarly, PARP
activity and PARP-1 content were not affected by NR (FIG. 26H).
Altogether, these results suggest that NR increases NAD.sup.+ by
direct NAD.sup.+ biosynthesis rather than by indirectly affecting
the major NAD.sup.+ salvage (Nampt) or consumption (PARPs)
pathways. Importantly, this increase in NAD.sup.+ was not linked to
changes in cellular glycolytic rates or ATP levels, which would be
expected if NAD.sup.+/NADH ratios had been altered to the point of
compromising basic cellular functions.
Example 16
NR Treatment Enhances SIRT1 and SIRT3 Activity
[0186] The ability of NR to increase intracellular NAD.sup.+ levels
both in vivo and in vitro prompted us to test whether it could
activate sirtuin enzymes. Confirming this hypothesis, NR
dose-dependently decreased the acetylation of FOXO1 in a
SIRT1-dependent manner (FIG. 27A). This deacetylation of FOXO1 by
SIRT1 upon NR treatment resulted in its transcriptional activation,
leading to higher expression of target genes, such as Gadd45,
Catalase, Sod1 and Sod2. The lack of changes in SIRT1 protein
levels upon NR treatment (FIG. 27A) suggests that NR increases
SIRT1 activity by enhancing NAD.sup.+ bioavailability. The higher
SIRT1 activity in NR-treated cells was supported by mRNA expression
analysis. Consistent with SIRT1 being a negative regulator of Ucp2
expression, NR decreased Ucp2 mRNA levels (FIG. 27B). Importantly,
knocking down Sirt1 prevented the action of NR on Ucp2 expression
(FIG. 27B). Similarly, the higher expression of a FOXO1 target
gene, Sod2, upon NR treatment was also prevented by the knockdown
of either Foxo1 or Sirt1 (FIG. 27B). This suggested that NR leads
to a higher Sod2 expression thought the activation of SIRT1, which
then deacetylates and activates FOXO1. Importantly, the knock-down
of SIRT1 did not compromise the ability of NR to increase
intracellular NAD.sup.+ content, indicating that NR uptake and
metabolism into NAD.sup.+ is not affected by SIRT1 deficiency (FIG.
27C).
[0187] In line with the increase in mitochondrial NAD.sup.+ levels
(FIG. 1E-F) and the potential consequent activation of
mitochondrial sirtuins, NR also reduced the acetylation status of
Ndufa9 and SOD2 (FIGS. 27D and 27E, respectively), both targets for
SIRT3 (Ahn et al., 2008; Qiu et al., 2010). SOD2 deacetylation has
been linked to a higher intrinsic activity. In line with these
observations, NR treatment enhanced SOD2 activity (FIG. 27E). To
ensure that NR-induced SOD2 deacetylation was consequent to SIRT3
activation, we used mouse embryonary fibroblast (MEFs) established
from SIRT3 KO mice. The absence of SIRT3 was reflected by the
higher basal acetylation of SOD2 (FIG. 27F). Importantly, NR was
unable to decrease the acetylation status of SOD2 in SIRT3.sup.-/-
MEFs (FIG. 27F), despite that NAD.sup.+ levels increased to similar
levels as in SIRT3.sup.+/+ MEFs (FIG. 27G). These results clearly
indicate that NR triggers SIRT3 activity, probably by increasing
mitochondrial NAD.sup.+ levels, inducing the concomitant
deacetylation of its mitochondrial targets. Strikingly, not all
sirtuins were affected by NR, as the acetylation of tubulin, a
target of the cytoplasmic SIRT2, was not altered.
Example 17
NR Supplementation Enhances Energy Expenditure
[0188] Given the promising role of sirtuins to protect against
metabolic disease, we next evaluated the effects of long-term NR
administration in vivo. We fed 10-week-old male C57Bl/6J mice with
either chow (CD) or high-fat diet (HFD), supplemented or not with
NR at 400 mg/kg/day. While NR had no effect on the body weight (BW)
on CD, HFD-induced body weight gain was significantly attenuated by
NR (FIG. 28A), due to reduced fat mass (FIG. 28B). This was visibly
translated into a significant lower weight of the epididymal depot
in NR-fed mice. Importantly, this was not due to redistribution of
lipids to other tissues, most notably to liver, which actually
contained 40% less triglycerides.
[0189] The reduced body weight gain of NR-fed mice upon HFD was not
due to reduced food intake, as NR-fed mice actually had a tendency
to eat more, especially on HFD conditions (FIG. 28C). Similarly, NR
did not affect the activity pattern of mice (FIG. 28D), indicating
that the lower BW on HFD was not consequent to different physical
activity. Rather, the phenotype was due to enhanced energy
expenditure (EE). Mice on CD had a marked tendency to display
higher O.sub.2 consumption rates when fed with NR, and this
tendency became clearly significant under HFD conditions (FIG.
28E). Of note, NR-fed mice became more flexible in their use of
energy substrates, as reflected in the higher amplitude of the
changes in RER between feeding and fasting periods in CD
conditions. Altogether, these results indicate that NR lowers
HFD-induced BW gain by enhancing EE.
[0190] From a metabolic perspective, NR- and vehicle-fed mice had
similar fasting blood glucose levels in either CD or HFD conditions
(FIG. 28F). However, fasting insulin levels were much lower in
NR-supplemented mice (FIG. 28G). This lower insulin/glucose ratio
is indicative of insulin sensitization after NR administration.
This speculation was further supported by glucose tolerance tests.
NR promoted a slight, albeit not significant, improvement in
glucose tolerance (FIG. 28H) in mice fed a HFD, accompanied by a
robust reduction in insulin secretion (FIG. 28I). Therefore, NR-fed
mice on HFD display a better glucose disposal with lower insulin
levels. In order to conclusively establish whether NR fed mice were
more insulin sensitive, we performed hyperinsulinemic-euglycemic
clamps on CD and CD-NR mice. We chose not to perform this analysis
on the HFD groups in order to avoid the possible influence of
differential BW. Mice supplemented with NR required an almost
2-fold higher glucose infusion rate to maintain euglycemia (FIG.
28J). This was consequent to a very marked increase in
insulin-induced muscle glucose uptake (FIG. 28J). This observation
unequivocally demonstrates that NR-fed mice are more
insulin-sensitive. Furthermore, NR partially prevented the increase
in total (FIG. 28K) and LDL cholesterol levels induced by HFD, even
though HDL-cholesterol levels were unaffected. The amelioration of
cholesterol profiles is fully in line with previous observations
from the use of other NAD.sup.+ precursors, such as NA.
Example: 18
NR Enhances the Oxidative Performance of Skeletal Muscle and Brown
Adipose Tissue
[0191] NR-fed mice had a clear tendency to display a better
endurance performance than vehicle fed mice. This tendency was
significantly accentuated upon HFD (FIG. 29A), suggesting an
enhanced muscle oxidative performance. Similarly, NR-fed mice, both
on CD and HFD, showed enhanced thermogenic capacity, as manifested
in the ability to maintain body temperature during cold exposure
(FIG. 29B). The latter observation hints toward an improvement in
brown adipose tissue (BAT) oxidative performance. To gain further
insight into the ability of BAT and muscle to enhance their
oxidative performance, we performed some histological analysis.
Gastrocnemius muscles from NR mice displayed a more intense SDH
staining than their vehicle-fed littermates, indicating a higher
oxidative profile. Electron microscopy revealed that mitochondria
in BAT of NR-fed mice, despite not being significantly larger, had
more abundant cristae (FIG. 29C), which has been linked to
increased respiratory capacity. Altogether, the above results
suggest that NR supplemented mice display a higher oxidative
capacity due to enhanced mitochondrial function.
Example 19
Chronic NR Feeding Increases NAD.sup.+ In Vivo in a Tissue-Specific
Manner
[0192] We next wondered how chronic NR feeding would affect
NAD.sup.+ metabolism in mice. Chronic NR supplementation increased
NAD.sup.+ levels in both CD and HFD (FIG. 30A) conditions in some
tissues, including liver and muscle, but not in others, such as
brain or white adipose tissue (WAT). Interestingly, NAD.sup.+ was
also higher in the BAT of NR-fed mice, but only on HFD (FIG. 30B
and. These differences could be due to the differential expression
of NRKs in tissues. NRKs initiate NR metabolism into NAD.sup.+.
There are two mammalian NRKs: NRK1 and NRK2. While we found NRK1
expressed ubiquitously, NRK2 was mainly present in cardiac and
skeletal muscle tissues, as previously described, but also
detectable in BAT and liver, in line with the better ability of
these tissues to respond to NR.
[0193] We also tested whether the increase in NAD.sup.+ would be
concomitant to changes in other NAD.sup.+ metabolites. Strikingly,
NADH and nicotinamide (NAM) levels were largely diminished in
muscles from NR-fed mice (FIG. 30B), indicating that NR
specifically increases NAD.sup.+, but not necessarily other
by-products of NAD.sup.+ metabolism. We analyzed in vivo whether
the activity of major NAD.sup.+ degrading enzymes or the levels of
Nampt could also contribute to the increase in NAD.sup.+ after
chronic NR supplementation. As previously observed in HEK293T cells
(FIG. 26G-H), PARP-1 levels and global PARylation were similar in
muscle (FIG. 30C) and livers from NR- and vehicle-fed mice,
indicating that the enhanced NAD.sup.+ content cannot be explained
by differential NAD.sup.+ consumption through PARP activity. Nampt
mRNA (FIG. 30D) and protein (FIG. 30C) levels were also similar in
NR and vehicle fed mice, suggesting that NAD.sup.+ salvage pathways
do not explain the differences in NAD.sup.+ levels. We furthermore
could not detect differences in mRNA expression of the different
NMN adenylyltransferase (NMNAT) enzymes (FIG. 30D). Altogether,
these results reinforce the notion that the higher NAD.sup.+ levels
observed in tissues from NR-fed mice is consequent to an increase
in direct NAD.sup.+ synthesis from NR.
Example 20
NR Enhances Sirtuin Activity In Vivo
[0194] Higher NAD.sup.+ levels were also accompanied by higher
sirtuin activity in vivo. A prominent deacetylation of SIRT1 and
SIRT3 targets (FOXO1 and SOD2, respectively) was observed in the
skeletal muscle, liver and BAT, where NAD.sup.+ content was induced
by NR, but not in brain and WAT, where NAD.sup.+ levels were
unaffected by NR supplementation (FIG. 31A). We also evaluated
PGC-1.alpha. acetylation as a second readout of SIRT1 activity. We
were unable to detect PGC-1.alpha. in total lysates from WAT or
brain, but in muscle, liver and BAT PGC-1.alpha. was deacetylated
upon NR treatment. These observations highlight how NR can only
induce sirtuin activity in tissues where NAD.sup.+ accumulates.
Like in cultured cells, we could not detect changes in the
acetylation status of the SIRT2 target tubulin, suggesting either
that increasing NAD.sup.+ might not affect the activity of all
sirtuins equally, that the increase is only compartment-specific or
that additional regulatory elements, like class I and II HDACs,
also contribute to tubulin acetylation status.
[0195] In line with the changes in acetylation levels of
PGC-1.alpha., a key transcriptional regulator of mitochondrial
biogenesis, we could observe either an elevated expression or a
strong tendency towards an increase (P<0.1) of nuclear genes
encoding transcriptional regulators of oxidative metabolism (Sirt1,
PGC-1.alpha.; mitochondrial transcription factor A (Tfam)) and
mitochondrial proteins (Mitofusin 2 (MX), Cytochrome C (Cyt C),
Medium Chain Acyl-coA Dehydrogenase (MCAD), Carnitine
palmitoyltransferase-1b (CPT-1b), Citrate Synthase (CS) or ATP
synthase lipid binding protein (ATP5g1)) in quadriceps muscles from
NR-fed mice (FIG. 31B). Conversely, in brain, where NAD.sup.+ and
sirtuin activity levels were not affected by NR feeding, the
expression of these genes was not altered (FIG. 31B). Consistently
also with enhanced mitochondrial biogenesis, mitochondrial DNA
content was increased in muscle, but not in brain from NR-fed mice
(FIG. 31C). Finally, mitochondrial protein content also confirmed
that mitochondrial function was only enhanced in tissues in which
NAD.sup.+ content was increased (FIG. 31D). This way, while muscle,
liver and BAT showed a prominent increase in mitochondrial proteins
(Complex V--ATP synthase subunit .alpha. and porin), such change
was not observed in brain or WAT. Altogether, these results suggest
that NR feeding increases mitochondrial biogenesis in a
tissue-specific manner, consistent with the tissue-specific nature
of the increases in NAD.sup.+ and sirtuin activity observed in
NR-fed mice. The higher number of mitochondria, together with the
different morphological mitochondrial profiles found in NR-fed mice
(FIG. 29C) would contribute to explain the higher oxidative
profile, energy expenditure and protection against metabolic damage
observed upon NR feeding.
Example 21
PARP Activity and NAD+ in Aged Mammals and Worms
[0196] The PARP proteins, with PARP1 and PARP2 representing the
main PARP activities in mammals, were classically described as DNA
repair proteins, but recent studies have linked these proteins to
metabolism. Furthermore, an association between PARPs and lifespan
has been postulated, but a causal role remained unclear. To
establish the role of PARPs in aging, we compared global PARylation
in young versus old mice (24 and 103 weeks). Both in liver and
muscle of aged mice, PARylation was markedly increased (FIG. 33A).
In line with the hypothesis that PARP proteins are prime NAD.sup.+
consumers, NAD.sup.+ levels were robustly decreased in older mice
(FIG. 33B). Changes in NAD+ are generally translated into alterated
SIRT1 activity. The lower NAD+ levels in aged mice were indeed
reflected in hyperacetylation of the SIRT1 substrate PGC1,
indicative of reduced SIRT1 activity (FIG. 33C). To evaluate the
possible contribution of PARP activity and NAD.sup.+ metabolism in
the aging process, we turned to C. elegans, where it is easier to
evaluate the impact of genetic or pharmacological manipulations on.
The aging-associated and NAD+ lifespan PARylation changes were
evolutionary conserved as PARylation was also markedly increased
with age in nematodes (FIG. 33D), and NAD+ levels were lower (FIG.
33E). Changes in PARylation and NAD+ were attenuated in worms in
which the PARP1 homolog--pme-1 (15)--was mutated (FIG. 33D-E). The
residual PARylation is consistent with the presence of a second
PARP gene, pme-2, the worm homolog of the less active PARP2
protein. We further analyzed the natural aging process in worms by
monitoring the accumulation of the aging-associated lipid
peroxidation product lipofuscin, which was robustly reduced in
pme-1 worms (FIG. 33 F). Together, these data suggest that
disturbance of the PARP/NAD.sup.+-signaling network in aging is
evolutionary conserved.
Example 22
Longevity in C. Elegans with Pme-1 Mutation or PARP Inhibition
[0197] We next aimed to determine the causal role of PARPs in
aging. Strikingly, pme-1 deficient worms displayed a 29% mean
lifespan extension (FIG. 34A, p<0.001, see Table 8 for
statistics). To inhibit PARP activity we also fed worms from eggs
until death with two chemically distinct pan-PARP inhibitors, i.e.
AZD2281 (KU59436, olaparib), or ABT-888 (veliparib), resulting in a
15-23% lifespan extension (FIG. 34B, p<0.001 for AZD2281,
p<0.05 for ABT-888; FIG. 37; Table 8). The lifespan of the pme-1
mutant was not further extended by AZD2281, confirming that pme-1
is the major worm PARP activity (FIG. 34C). Consistent with the
hypothesis of NAD+ as a possible mediator of these effects, both
deletion of pme-1 gene or pharmacological PARP inhibition
significantly NAD+ increased levels (FIG. 34D). Although the role
of SIRT1 or its homologs in lifespan extension under basal,
unstressed, conditions is subject of intense debate, it holds a
central position in healthspan regulation in the context of disease
or cellular stress. Given the NAD+ dependence of SIRT1, we analyzed
epistasis by treating sir-2.1(ok434) mutant worms with AZD2281. In
this context, we lost the AZD2281-induced longevity in the
sir-2.1(ok434) mutant (FIG. 34E), confirming sir-2.1 dependence of
the lifespan extension.
Example 23
PARP Inhibition Increases Mitochondrial Function and ROS
Defense
[0198] Consistent with delayed aging, and in line with the data in
pme-1 mutants, AZD2281 reduced lipofuscin accumulation (FIG. 35A).
As NAD+ and SIRT1 are thought to influence oxidative metabolism, we
functionally characterized mitochondrial activity in
AZD2281-treated worms by measuring oxygen consumption rates. At day
3 of adulthood, AZD2281 robustly increased respiration (FIG. 35B),
in line with increased expression of citrate synthase (cts-1) and
cytochrome c oxidase subunit 4 (cox-4) (FIG. 35C). By using
confocal microscopy in the pmyo-3::mito::GFP reporter, which
expresses mitochondria-targeted GFP in the muscle, we also observed
a more intense fluorescence signal after AZD2281, indicating a more
dense mitochondrial network (FIG. 35D-E).
[0199] As changes in mitochondrial metabolism can cause oxidative
stress, we measured reactive oxygen species (ROS) during early
adulthood using the mitoSOX probe, which indicates specific
mitochondrial superoxide production. At day 3, ROS production was
decreased in AZD2281-treated worms, paralleled with a marked
induction of the mitochondrial antioxidant gene, sod-3 (FIG.
35F-G). The best-characterized transcriptional regulator of
antioxidant defense is daf-16, the C. elegans FOXO3A homolog.
AZD2281 failed to increase lifespan in daf-16 mutants (FIG. 351),
suggesting that the induction of antioxidant defense is key for
AZD2281 to grant longevity. Of relevance, FOXO3A has been described
as a deacetylation target for SIRT1 in mammals, and daf-16 was
shown to interact with sir-2. AZD2281 did not change expression of
daf-16 (FIG. 35H), in line with its reported regulation by
subcellular distribution rather than transcription.
Example 24
Supplementation of C. Elegans with the NAD+ Precursor NR Mimics the
Metabolic Lifespan Effects of PARP Mutation or Inhibition
[0200] In addition to their role in NAD+ metabolism, PARP proteins
could in theory also impact longevity through PARylation of
specific proteins. To confirm our hypothesis that it is indeed the
increase in NAD+ that induces longevity upon PARP inhibition, we
analyzed the impact of raising NAD+ levels through providing worms
with different NAD+ precursors. We focused on NAD+ the salvage
pathway precursors, nicotinamide (NAM) and NAM riboside (NR). NAM
is the end-product of the sirtuin and PARP reaction, whereas NR is
a recently discovered vitamin--both can serve as precursors of NAD
(re-)synthesis. Consistent with their function as NAD+ precursors,
NAM and NR increased NAD+ levels (FIG. 36A, FIG. S39A). Strikingly,
treatment of C. elegans with these NAD+ precursors also extended
lifespan (FIG. 36B, FIG. 38, FIG. 39B, see Table 8 for statistics)
in a sir-2.1-dependent fashion (FIG. 36C).
[0201] As was observed for AZD2281, NR and NAM also increase muscle
mitochondrial content (FIG. 36D, FIG. 39E) and affect mitochondrial
morphology (FIG. 36E). Similarly, both NAM and NR increased
respiration at day 3 (FIG. 36F, FIG. 39C) with a trend for
increased cts-1 expression (FIG. 36G, FIG. S3C), and significantly
higher sod-3 expression, without affecting daf-16 mRNA levels (FIG.
36H, FIG. 39D), thereby mimicking the effects of PARP inhibition on
lifespan and mitochondrial metabolism. Importantly, the lifespan
extension of NR was also completely abolished in daf-16 mutant
worms (FIG. 361). The fact that two independent strategies to boost
NAD+ levels promote a similar phenotype strengthens the hypothesis
that NAD+ might be a critical metabolite influencing mitochondrial
fitness and lifespan in a sir2.1-dependent way.
[0202] To summarize, NAD+ our data indicate that PARP activity, by
modulating availability, plays an important role to preserve
(mitochondrial) fitness. Not only is PARylation increased and are
NAD+ levels reduced in aged worms and mice, but we also show that
interventions aimed to safeguard NAD+ levels curb the aging process
and extend lifespan in C. elegans. Furthermore, our data also
provide evidence for a role of sir-2.1 in lifespan regulation, at
least in the context of increased availability of its substrate
NAD+. We hence propose a model in which elevation of NAD+ levels
activate sir-2.1, resulting in the deacetylation of daf-16, which
improves oxidative stress defense through sod-3 (FIG. 36J). The
fact that PARP inhibitors and NAD+ precursors also increase NAD+
levels in mammals suggests that these beneficial metabolic effects
may also apply to humans and warrants further validation.
TABLE-US-00001 TABLE 1 qRT-PCR primers for quantification of gene
expression Gene Primers ACC1 5'-GAC AGA CTG ATC GCA GAG AAA G-3'
5'-TGG AGA GCC CCA CAC ACA-3' ACC2 5'-CCC AGC CGA GTT TGT CAC T-3'
5'-GGC GAT GAG CAC CTT CTC TA-3' ACO 5'-CCC AAC TGT GAC TTC CAT
T-3' 5'-GGC ATG TAA CCC GTA GCA CT-3' ATP5g1 5'-GCT GCT TGA GAG ATG
GGT TC-3' 5'-AGT TGG TGT GGC TGG ATC A-3' COX17 5'-CGT GAT GCG TGC
ATC ATT GA-3' 5'-CAT TCA CAA AGT AGG CCA CC-3' Cyclophyllin 5'-TGG
AGA GCA CCA AGA CAG ACA -3' B 5'-TGC CGG AGT CGA CAA TGA T-3'
Cytochrome 5'-TCC ATC AGG GTA TCC TCT CC-3' C 5'-GGA GGC AAG CAT
AAG ACT GG-3' Dio2 5'-GCA CGT CTC CAA TCC TGA AT-3' 5'-TGA ACC AAA
GTT GAC CAC CA -3' ERR 5'-ACTGCCACTGCAGGATGAG-3'
5'-CACAGCCTCAGCATCTTCAA-3' GK 5'-ACA TTG TGC GCC GTG CCT GTG AA-3'
5'-AGC CTG CGC ACA CTG GCG TGA AA-3' G6Pase 5'-CCG GAT CTA CCT TGC
TGC TCA CTT T-3' 5'-TAG CAG GTA GAA TCC AAG CGC GAA AC-3' Malic
5'-GCC GGC TCT ATC CTC CTT TG-3' enzyme 5'-TTT GTA TGC ATC TTG CAC
AAT CTT T-3' MCD 5'-TGG ATG GCT GAC AGC AGC CTC AA-3' 5'-CTG AGG
ATC TGC TCG GAA GCT TTG-3' MCAD 5'-GAT CGC AAT GGG TGC TTT TGA TAG
AA-3' 5'-AGC TGA TTG GCA ATG TCT CCA GCA AA-3' mCPT1 5'-TTG CCC TAC
AGC TGG CTC ATT TCC -3' 5'-GCA CCC AGA TGA TTG GGA TAC TGT-3'
Myosin 1 5'-GAG TAG CTC TTG TGC TAC CCA GC -3' 5'-AAT TGC TTT ATT
CTG CTT CCA CC -3' Myosin 2A 5'-GCA AGA AGC AGA TCC AGA AAC-3'
5'-GGT CTT CTT CTG TCT GGT AAG TAA GC -3' Myosin 2X 5'-GCA ACA GGA
GAT TTC TGA CCT CAC-3' 5'-CCA GAG ATG CCT CTG CTT C-3' Ndufa2
5'-GCA CAC ATT TCC CCA CAC TG-3' 5'-CCC AAC CTG CCC ATT CTG AT-3'
Ndufb3 5'-TAC CAC AAA CGC AGC AAA CC-3' 5'-AAG GGA CGC CAT TAG AAA
CG-3' Ndufb5 5'-CTT CGA ACT TCC TGC TCC TT-3' 5'-GGC CCT GAA AAG
AAC TAC G-3' PARP-1 5'-GGA GCT GCT CAT CTT CAA CC-3' 5'-GCA GTG ACA
TCC CCA GTA CA-3' PARP-2 5'-GGA AGG CGA GTG CTA AAT GAA-3' 5'-AAG
GTC TTC ACA GAG TCT CGA TTG-3' PARP-3 5'-CCT GCT GAT AAT CGG GTC
AT-3' 5'-TTG TTG TTG TTG CCG ATG TT-3' PARP-4 5'-GTT AAA TTT TGC
ACT CCT GGA G-3' 5'-AAT GTG AAC ACT GTC AAG AGG AAC A-3' PARP-5a
5'-TAG AGG CAT CGA AAG CTG GT-3' 5'-CAG GCA TTG TGA AGG GG-3'
PARP-5b 5'-GGC CCT GCT TAC ACC ATT G-3' 5'-CGT GCT TGA CCA GAA GTT
CA-3' PARP-6 5'-TTT CCA GCC ATC GAA TAA GG-3' 5'-ACC ACT TGC CTT
GAA CCA AC-3' PARP-7 5'-AAA ACC CCT GGA AAT CAA CC-3' 5'-AGA AGG
ATG CGC TTC TGG TA-3' PARP-8 5'-TCC ACC ATT AAA TCG CAC AA-3'
5'-GCT CCA TTT TCG ATG TCT TG-3' PARP-9 5'-ACC TGA AGA ATG GCC TAT
TAC ATG G-3' 5'-ACA GCT CAG GGT AGA GAT GC-3' PARP-10 5'-CAA GAT
CCT GCA GAT GCA AA-3' 5'-TTG GAG AAG CAC ACG TTC TG-3' PARP-11
5'-CAA TGA GCA GAT GCT ATT TCA TG-3' 5'-CAC CAA TTA GCA CTC GAG
CA-3' PARD-12 5'-CGG ATC CAG AAC ATG GGC-3' 5'-GGC ATC TCT CGC AAA
GTA GC-3' PARP-14 5'-GGC AAA CGC AAT GGA ACT AT-3' 5'-AGC ACG TTC
CTA AGC CTT GA-3' PARP-16 5'-CCG TGT GCC TTA TGG AAA CT-3' 5'-TGG
ATT GTG TCT GGG CAC-3' PDK4 5'-AAA GGA CAG GAT GGA AGG AAT CA-3'
5'-ATT AAC TGG CAG AGT GGC AGG TAA-3' PEPCK 5'-CCA CAG CTG CTG CAG
AAC A-3' 5'-GAA GGG TCG CAT GGC AAA-3' PGC-1 5'-AAG TGT GGA ACT CTC
TGG AAC TG-3' 5'-GGG TTA TCT TGG TTG GCT TTA TG-3' PPAR 5'-CCT GAA
CAT CGA GTG TCG AAT AT-3' 5'-GGT TCT TCT TCT GAA TCT TGC AGC T-3'
SREBP1 5'-GGC CGA GAT GTG CGA ACT-3' 5'-TTG TTG ATG AGC TGG AGC ATG
T-3' Troponin 5'-CCA GCA CCT TCA GCT TCA GGT CCT TGA T-3' I 5'-TGC
CGG AAG TTG AGA GGA AAT CCA AGA T-3' UCP1 5'-GGC CCT TGT AAA CAA
CAA AAT AC-3' 5'-GGC AAC AAG AGC TGA CAG TAA AT-3' UCP2 5'-TGG CAG
GTA GCA CCA CAG G-3' 5'-CAT CTG GTC TTG CAG CAA CTC T-3' UCP3
5'-ACT CCA GCG TCG CCA TCA GGA TTC T-3' 5'-TAA ACA GGT GAG ACT CCA
GCA ACT T-3'
TABLE-US-00002 TABLE 2 Primers for mtDNA determination. mtDNA
specific 5'-CCG CAA GGG AAA GAT GAA AGA C-3' (murine) 5'-TCG TTT
GGT TTC GGG GTT TC-3' nuclear specific 5'-GCC AGC CTC TCC TGA TTT
TAG TGT-3' (murine) 5'-GGG AAC ACA AAA GAC CTC TTC TGG-3' mtDNA
specific 5'-CTA TGT CGC AGT ATC TGT CTT TG-3' (human) 5'-GTT ATG
ATG TCT GTG TGG AAA G-3' nuclear specific 5'-GTT TGT GTG CTA TAG
ATG ATA TTT TAA ATT (human) G-3' 5'-CAT TAA ACA GTC TAC AAA ACA
TAT-3'
TABLE-US-00003 TABLE 3 ChIP primers PDK4 5'-AAC CCT CCT CCC TCT CAC
CCT-3' 5'-ACA CCA ATC AGC TCA GAG AA-3' UCP-3 5'-GAA TGT CAG GCC
TCT AAG AA-3' 5'-CAG GAG GTG TGT GAC AGC AT-3'
TABLE-US-00004 TABLE 4 RT-qPCR primers for quantification of gene
expression. Gene Primers ACO CCC AAC TGT GAC TTC CAT T GGC ATG TAA
CCC GTA GCA CT ATP5g1 GCT GCT TGA GAG ATG GGT TC AGT TGG TGT GGC
TGG ATC A COX17 CGT GAT GCG TGC ATC ATT GA CAT TCA CAA AGT AGG CCA
CC Citrate GGA GCC AAG AAC TCA TCC TG TCT GGC CTG CTC CTT AGG TA
Cyclo- TGG AGA GCA CCA AGA CAG ACA phyl- TGC CGG AGT CGA CAA TGA T
lin B Cyto TCC ATC AGG GTA TCC TCT CC chrome GGA GGC AAG CAT AAG
ACT GG C Dio2 GCA CGT CTC CAA TCC TGA AT TGA ACC AAA GTT GAC CAC CA
ERR ACT GCC ACT GCA GGA TGA G CAC AGC CTC AGC ATC TTC AA FOXO1 AAG
GAT AAG GGC GAC AGC AA TCC ACC AAG AAC TCT TTC CA G6Pase CCG GAT
CTA CCT TGC TGC TCA CTT T TAG CAG GTA GAA TCC AAG CGC GAA AC GK ACA
TTG TGC GCC GTG CCT GTG AA AGC CTG CGC ACA CTG GCG TGA AA Kir6.2
CTG TCC CGA AAG GGC ATT AT CGT TGCAGT TGC CTT TCT TG Insulin GTG
GGG AGC GTG GCT TCT TCT A ACT GAT CCA CAA TGC CAC GCT TCT Insulin
CGA GTG CCC GTC TGG CTA TA receptor GGC AGG GTCCCA GAC ATG LCAD GTA
GCT TAT GAA TGT GTG CAA CTC GTC TTG CGA TCA GCT CTT TCA TTA L-CPT1
GCA CTG CAG CTC GCA CAT TAC AA CTC AGA CAG TAC CTC CTT CAG GAA A
MCD TGG ATG GCT GAC AGC AGC CTC AA CTG AGG ATC TGC TCG GAA GCT TTG
MCAD GAT CGC AAT GGG TGC TTT TGA TAG AA AGC TGA TTG GCA ATG TCT CCA
GCA AA mCPT1 TTG CCC TAC AGC TGG CTC ATT TCC GCA CCC AGA TGA TTG
GGA TAC TGT Ndufa2 GCA CAC ATT TCC CCA CAC TG CCC AAC CTG CCC ATT
CTG AT PEPCK CCACAG CTG CTG CAGAACA GAAGGG TCG CAT GGCAAA PPAR AGG
AAG CCG TTC TGT GAC AT TTG AAG GAG CTT TGG GAA GA PC AGG GGC TGC
TGT TGA TGG AC CAG GGG CAC TCG TAC AGG AAG C PDK4 AAA GGA CAG GAT
GGA AGG AAT CA ATT AAC TGG CAG AGT GGC AGG TAA SDH GAA CTG CAC ACA
GAC CTG C GAC TGG GTT AAG CCA ATG CTC SIRT1 TGT GAA GTT ACT GCA GGA
GTG TAA A GCA TAG ATA CCG TCT CTT GAT CTG AA SIRT3 AGG TGG AGG AAG
CAG TGA GA GCT TGG GGT TGT GAA AGA AA PDX1 AAT CCA CCA AAG CTC ACG
CGT GGA A TGA TGT GTC TCT CGG TCA AGT TCA A PGC-1 AAG TGT GGA ACT
CTC TGG AAC TG GGG TTA TCT TGG TTG GCT TTATG PGC-1 TGG AGA CTG CTC
TGG AAG GT TGC TGC TGT CCT CAA ATA CG TFAm CCA AAA AGA CCT CGT TCA
GC ATG TCT CCG GAT CGT TTC AC Troponin CCA GCA CCT TCA GCT TCA GGT
CCT TGA T I TGC CGG AAG TTG AGA GGA AAT CCA AGA T UCP1 GGC CCT TGT
AAA CAA CAA AAT AC GGC AAC AAG AGC TGA CAG TAA AT UCP2 ACC AAG GGC
TCA GAG CAT GCA TGG CTT TCA GGA GAG TAT CTT TG UCP3 ACT CCA GCG TCG
CCA TCA GGA TTC T TAA ACA GGT GAG ACT CCA GCA ACT T
TABLE-US-00005 TABLE 5 Primers for mtDNA determination. mtDNA
5'-CCG CAA GGG AAA GAT GAA AGA specific C-3' (murine) 5'-TCG TTT
GGT TTC GGG GTT nuclear 5'-GCC AGC CTC TCC TGA TTT TAG specific
TGT-3' (murine) 5'-GGG AAC ACA AAA GAC CTC TTC TGG-3'
TABLE-US-00006 TABLE 6 Primers for ChIP SIRT1- 5'-TCC CGC AGC CGA
GCC GCG GGG-3' 91 5'-TCT TCC AAC TGC CTC TCT GGC CCT CCG -3' human
5'-CAT TTC TCC ACC TCA CTG AAA K19 CTG-3' 5'-AAT GTG TTA GTG CAT
GCA murine 5'-AAG GGT GGA GGT GTC TTG K19 GT-3' 5'-GCT TCT TTA CAC
TCC
TABLE-US-00007 TABLE 7 RT-qPCR primers for quantification of gene
expression. Gene Forward Reverse sod-3 CTAAGGATGGTGGAGAACCTTCA
CGCGCTTAATAGTGTCCATCAG (C08A9.1) daf-16 ATCCAATTGTGCCAAGCACTAA
CCACCATTTTGATAGTTTCCATAGG (R13H8.1) cts-1 CTCGACAACTTCCCAGATAACC
GGTACAGGTTGCGATAGATGATAGC (T20G5.1) cox-4 GCCCCAATTCGCGCCAAGGA
AGGTTGGCGGCAGTTCTGGG (W09C5.8) rrn-1.1 TTCTTCCATGTCCGGGATAG
CCCCACTCTTCTCGAATCAG (F31C3.7) act-1 GCTGGACGTGATCTTACTGATTACC
GTAGCAGAGCTTCTCCTTGATGTC (T04C12.6)
TABLE-US-00008 TABLE 8 Summary of lifespan experiments variation
P-values mean lifespan .+-. compared to against strains and culture
conditions SE (days) control (%) control N(trials) N2 pme- 19.7
.+-. 0.61 96/24 (2) 1(ok988) 25.5 .+-. 0.52 +29.4 <10.sup.-3
91/29 (2) N2 + water 20.1 .+-. 0.59 138/42 (3) N2 + AZD 100 nM 24.7
.+-. 0.55 +22.9 <10.sup.-3 141/39 (3) N2 + water 19.4 .+-. 0.98
55/5 (1) N2 + ABT 100 nM 22.3 .+-. 1.01 +15.0 0.05 52/8 (1) N2 +
water 19.8 .+-. 0.87 53/7 (1) pme-1(ok988) + water 27.2 .+-. 0.55
+37.4 <10.sup.-3 49/11 (1) N2 + AZD 100 nM 22.8 .+-. 0.9 +15.2
0.02 48/12 (1) pme-1(ok988) + AZD 100 nM 26.1 .+-. 0.78 +31.2 .sup.
ns.sup.a 49/11 (1) N2 + water 20.5 .+-. 0.737 83/37 (2)
sir-2.1(ok434) + water 22.9 .+-. 0.624 +11.7 ns 88/32 (2) N2 + AZD
100 nM 24.7 .+-. 0.669 +20.5 <10.sup.-3 89/31 (2) sir-2.1(ok434)
+ AZD 100 nM 21.9 .+-. 0.528 +6.7 .sup. ns.sup.b 84/36 (2) N2 +
water 21.7 .+-. 1.24 30/30 (1) daf-16(mu86) + water 17.5 .+-. 0.69
-19.3 0.0009 36/24 (1) N2 + AZD 100 nM 26.5 .+-. 0.77 +22.1 0.004
41/19 (1) daf-16(mu86) + AZD 100 nM 18.2 .+-. 0.76 -16.1 .sup.
ns.sup.c 37/23 (1) N2 + water 19.5 .+-. 0.51 188/52 (4) N2 + NR 500
1iM 23.5 .+-. 0.56 +20.1 <10.sup.-3 172/68 (4) N2 + water 20.7
.+-. 0.80 79/41 (2) sir-2.1(ok434) + water 23.4 .+-. 0.67 +13.0 ns
80/40 (2) N2 + NR 500 1iM 24.9 .+-. 0.68 +20.3 0.0008 86/34 (2)
sir-2.1(ok434) + NR 500 1iM 23.2 .+-. 0.61 +12.0 .sup. ns.sup.b
76/44 (2) N2 + water 20.7 .+-. 0.80 79/41 (2) daf-16(mu86) + water
17.1 .+-. 0.47 -17.4 <10.sup.-3 76/44 (2) N2 + NR 500 1iM 24.9
.+-. 0.68 +20.3 0.0008 86/34 (2) daf-16(mu86) + NR 500 1iM 19.1
.+-. 0.51 -7.8 0.01.sup.c 88/32 (2) N2 + water 19.9 .+-. 0.95 54/6
(1) N2 + NAM 200 1iM 23.5 .+-. 1.03 +18.1 0.01 50/10 (1) N2 + NAM
500 1iM 22.6 .+-. 0.92 +13.5 ns 52/8 (1) N2 + NAM 1000 1iM 21.8
.+-. 0.85 +9.5 ns 46/14 (1) N2 + water 19.4 .+-. 0.97 55/5 (1) N2 +
AZD 100 nM 24.5 .+-. 0.94 +25.6 0.001 52/8 (1) N2 + AZD 500 nM 22.2
.+-. 1.15 +14.4 0.03 48/12 (1) N2 + AZD 1000 nM 21.2 .+-. 0.96 +9.2
ns 53/7 (1) N2 + water 19.4 .+-. 0.97 55/5 (1) N2 + ABT 100 nM 22.5
.+-. 1.05 +16.0 0.04 52/8 (1) N2 + ABT 500 nM 22.1 .+-. 1.20 +13.9
ns 49/11 (1) N2 + ABT 1000 nM 20.0 .+-. 1.23 +3.6 ns 50/10 (1) N2 +
water 17.9 .+-. 0.84 54/6 (1) N2 + NR 200 1iM 21.1 .+-. 0.88 +17.9
0.04 44/16 (1) N2 + NR 500 1iM 20.6 .+-. 1.02 +15.1 0.04 43/17 (1)
N2 + NR 1000 1iM 20.0 .+-. 0.89 +11.7 ns 50/10 (1)
Other Embodiments
[0203] While the invention has been described in conjunction with
the detailed description thereof, the foregoing description is
intended to illustrate and not limit the scope of the invention,
which is defined by the scope of the appended claims. Other
aspects, advantages, and modifications are within the scope of the
following claims.
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Sequence CWU 1
1
162122DNAArtificial SequenceSynthesized primer 1gacagactga
tcgcagagaa ag 22218DNAArtificial SequenceSynthesized primer
2tggagagccc cacacaca 18319DNAArtificial SequenceSynthesized primer
3cccagccgag tttgtcact 19420DNAArtificial SequenceSynthesized primer
4ggcgatgagc accttctcta 20519DNAArtificial SequenceSynthesized
primer 5cccaactgtg acttccatt 19620DNAArtificial SequenceSynthesized
primer 6ggcatgtaac ccgtagcact 20720DNAArtificial
SequenceSynthesized primer 7gctgcttgag agatgggttc
20819DNAArtificial SequenceSynthesized primer 8agttggtgtg gctggatca
19920DNAArtificial SequenceSynthesized primer 9cgtgatgcgt
gcatcattga 201020DNAArtificial SequenceSynthesized primer
10cattcacaaa gtaggccacc 201121DNAArtificial SequenceSynthesized
primer 11tggagagcac caagacagac a 211219DNAArtificial
SequenceSynthesized primer 12tgccggagtc gacaatgat
191320DNAArtificial SequenceSynthesized primer 13tccatcaggg
tatcctctcc 201420DNAArtificial SequenceSynthesized primer
14ggaggcaagc ataagactgg 201520DNAArtificial SequenceSynthesized
primer 15gcacgtctcc aatcctgaat 201620DNAArtificial
SequenceSynthesized primer 16tgaaccaaag ttgaccacca
201719DNAArtificial SequenceSynthesized primer 17actgccactg
caggatgag 191820DNAArtificial SequenceSynthesized primer
18cacagcctca gcatcttcaa 201923DNAArtificial SequenceSynthesized
primer 19acattgtgcg ccgtgcctgt gaa 232023DNAArtificial
SequenceSynthesized primer 20agcctgcgca cactggcgtg aaa
232125DNAArtificial SequenceSynthesized primer 21ccggatctac
cttgctgctc acttt 252226DNAArtificial SequenceSynthesized primer
22tagcaggtag aatccaagcg cgaaac 262320DNAArtificial
SequenceSynthesized primer 23gccggctcta tcctcctttg
202425DNAArtificial SequenceSynthesized primer 24tttgtatgca
tcttgcacaa tcttt 252523DNAArtificial SequenceSynthesized primer
25tggatggctg acagcagcct caa 232624DNAArtificial SequenceSynthesized
primer 26ctgaggatct gctcggaagc tttg 242726DNAArtificial
SequenceSynthesized primer 27gatcgcaatg ggtgcttttg atagaa
262826DNAArtificial SequenceSynthesized primer 28agctgattgg
caatgtctcc agcaaa 262924DNAArtificial SequenceSynthesized primer
29ttgccctaca gctggctcat ttcc 243024DNAArtificial
SequenceSynthesized primer 30gcacccagat gattgggata ctgt
243123DNAArtificial SequenceSynthesized primer 31gagtagctct
tgtgctaccc agc 233223DNAArtificial SequenceSynthesized primer
32aattgcttta ttctgcttcc acc 233321DNAArtificial SequenceSynthesized
primer 33gcaagaagca gatccagaaa c 213426DNAArtificial
SequenceSynthesized primer 34ggtcttcttc tgtctggtaa gtaagc
263524DNAArtificial SequenceSynthesized primer 35gcaacaggag
atttctgacc tcac 243619DNAArtificial SequenceSynthesized primer
36ccagagatgc ctctgcttc 193720DNAArtificial SequenceSynthesized
primer 37gcacacattt ccccacactg 203820DNAArtificial
SequenceSynthesized primer 38cccaacctgc ccattctgat
203920DNAArtificial SequenceSynthesized primer 39taccacaaac
gcagcaaacc 204020DNAArtificial SequenceSynthesized primer
40aagggacgcc attagaaacg 204120DNAArtificial SequenceSynthesized
primer 41cttcgaactt cctgctcctt 204219DNAArtificial
SequenceSynthesized primer 42ggccctgaaa agaactacg
194320DNAArtificial SequenceSynthesized primer 43ggagctgctc
atcttcaacc 204420DNAArtificial SequenceSynthesized primer
44gcagtgacat ccccagtaca 204521DNAArtificial SequenceSynthesized
primer 45ggaaggcgag tgctaaatga a 214624DNAArtificial
SequenceSynthesized primer 46aaggtcttca cagagtctcg attg
244720DNAArtificial SequenceSynthesized primer 47cctgctgata
atcgggtcat 204820DNAArtificial SequenceSynthesized primer
48ttgttgttgt tgccgatgtt 204922DNAArtificial SequenceSynthesized
primer 49gttaaatttt gcactcctgg ag 225025DNAArtificial
SequenceSynthesized primer 50aatgtgaaca ctgtcaagag gaaca
255120DNAArtificial SequenceSynthesized primer 51tagaggcatc
gaaagctggt 205217DNAArtificial SequenceSynthesized primer
52caggcattgt gaagggg 175319DNAArtificial SequenceSynthesized primer
53ggccctgctt acaccattg 195420DNAArtificial SequenceSynthesized
primer 54cgtgcttgac cagaagttca 205520DNAArtificial
SequenceSynthesized primer 55tttccagcca tcgaataagg
205620DNAArtificial SequenceSynthesized primer 56accacttgcc
ttgaaccaac 205720DNAArtificial SequenceSynthesized primer
57aaaacccctg gaaatcaacc 205820DNAArtificial SequenceSynthesized
primer 58agaaggatgc gcttctggta 205920DNAArtificial
SequenceSynthesized primer 59tccaccatta aatcgcacaa
206020DNAArtificial SequenceSynthesized primer 60gctccatttt
cgatgtcttg 206125DNAArtificial SequenceSynthesized primer
61acctgaagaa tggcctatta catgg 256220DNAArtificial
SequenceSynthesized primer 62acagctcagg gtagagatgc
206320DNAArtificial SequenceSynthesized primer 63caagatcctg
cagatgcaaa 206420DNAArtificial SequenceSynthesized primer
64ttggagaagc acacgttctg 206523DNAArtificial SequenceSynthesized
primer 65caatgagcag atgctatttc atg 236620DNAArtificial
SequenceSynthesized primer 66caccaattag cactcgagca
206718DNAArtificial SequenceSynthesized primer 67cggatccaga
acatgggc 186820DNAArtificial SequenceSynthesized primer
68ggcatctctc gcaaagtagc 206920DNAArtificial SequenceSynthesized
primer 69ggcaaacgca atggaactat 207020DNAArtificial
SequenceSynthesized primer 70agcacgttcc taagccttga
207120DNAArtificial SequenceSynthesized primer 71ccgtgtgcct
tatggaaact 207218DNAArtificial SequenceSynthesized primer
72tggattgtgt ctgggcac 187323DNAArtificial SequenceSynthesized
primer 73aaaggacagg atggaaggaa tca 237424DNAArtificial
SequenceSynthesized primer 74attaactggc agagtggcag gtaa
247519DNAArtificial SequenceSynthesized primer 75ccacagctgc
tgcagaaca 197618DNAArtificial SequenceSynthesized primer
76gaagggtcgc atggcaaa 187723DNAArtificial SequenceSynthesized
primer 77aagtgtggaa ctctctggaa ctg 237823DNAArtificial
SequenceSynthesized primer 78gggttatctt ggttggcttt atg
237923DNAArtificial SequenceSynthesized primer 79cctgaacatc
gagtgtcgaa tat 238025DNAArtificial SequenceSynthesized primer
80ggttcttctt ctgaatcttg cagct 258118DNAArtificial
SequenceSynthesized primer 81ggccgagatg tgcgaact
188222DNAArtificial SequenceSynthesized primer 82ttgttgatga
gctggagcat gt 228328DNAArtificial SequenceSynthesized primer
83ccagcacctt cagcttcagg tccttgat 288428DNAArtificial
SequenceSynthesized primer 84tgccggaagt tgagaggaaa tccaagat
288523DNAArtificial SequenceSynthesized primer 85ggcccttgta
aacaacaaaa tac 238623DNAArtificial SequenceSynthesized primer
86ggcaacaaga gctgacagta aat 238719DNAArtificial SequenceSynthesized
primer 87tggcaggtag caccacagg 198822DNAArtificial
SequenceSynthesized primer 88catctggtct tgcagcaact ct
228925DNAArtificial SequenceSynthesized primer 89actccagcgt
cgccatcagg attct 259025DNAArtificial SequenceSynthesized primer
90taaacaggtg agactccagc aactt 259122DNAArtificial
SequenceSynthesized primer 91ccgcaaggga aagatgaaag ac
229220DNAArtificial SequenceSynthesized primer 92tcgtttggtt
tcggggtttc 209324DNAArtificial SequenceSynthesized primer
93gccagcctct cctgatttta gtgt 249424DNAArtificial
SequenceSynthesized primer 94gggaacacaa aagacctctt ctgg
249523DNAArtificial SequenceSynthesized primer 95ctatgtcgca
gtatctgtct ttg 239622DNAArtificial SequenceSynthesized primer
96gttatgatgt ctgtgtggaa ag 229731DNAArtificial SequenceSynthesized
primer 97gtttgtgtgc tatagatgat attttaaatt g 319824DNAArtificial
SequenceSynthesized primer 98cattaaacag tctacaaaac atat
249921DNAArtificial SequenceSynthesized primer 99aaccctcctc
cctctcaccc t 2110020DNAArtificial SequenceSynthesized primer
100acaccaatca gctcagagaa 2010120DNAArtificial SequenceSynthesized
primer 101gaatgtcagg cctctaagaa 2010220DNAArtificial
SequenceSynthesized primer 102caggaggtgt gtgacagcat
2010320DNAArtificial SequenceSynthesized primer 103ggagccaaga
actcatcctg 2010420DNAArtificial SequenceSynthesized primer
104tctggcctgc tccttaggta 2010520DNAArtificial SequenceSynthesized
primer 105aaggataagg gcgacagcaa 2010620DNAArtificial
SequenceSynthesized primer 106tccaccaaga actctttcca
2010720DNAArtificial SequenceSynthesized primer 107ctgtcccgaa
agggcattat 2010820DNAArtificial SequenceSynthesized primer
108cgttgcagtt gcctttcttg 2010922DNAArtificial SequenceSynthesized
primer 109gtggggagcg tggcttcttc ta 2211024DNAArtificial
SequenceSynthesized primer 110actgatccac aatgccacgc ttct
2411120DNAArtificial SequenceSynthesized primer 111cgagtgcccg
tctggctata 2011218DNAArtificial SequenceSynthesized primer
112ggcagggtcc cagacatg 1811324DNAArtificial SequenceSynthesized
primer 113gtagcttatg aatgtgtgca actc 2411424DNAArtificial
SequenceSynthesized primer 114gtcttgcgat cagctctttc atta
2411523DNAArtificial SequenceSynthesized primer 115gcactgcagc
tcgcacatta caa 2311625DNAArtificial SequenceSynthesized primer
116ctcagacagt acctccttca ggaaa 2511720DNAArtificial
SequenceSynthesized primer 117aggaagccgt tctgtgacat
2011820DNAArtificial SequenceSynthesized primer 118ttgaaggagc
tttgggaaga 2011920DNAArtificial SequenceSynthesized primer
119aggggctgct gttgatggac 2012022DNAArtificial SequenceSynthesized
primer 120caggggcact cgtacaggaa gc 2212123DNAArtificial
SequenceSynthesized primer 121aaaggacagg atggaaggaa tca
2312224DNAArtificial SequenceSynthesized primer 122attaactggc
agagtggcag gtaa 2412319DNAArtificial SequenceSynthesized primer
123gaactgcaca cagacctgc 1912421DNAArtificial SequenceSynthesized
primer 124gactgggtta agccaatgct c 2112525DNAArtificial
SequenceSynthesized primer 125tgtgaagtta ctgcaggagt gtaaa
2512626DNAArtificial SequenceSynthesized primer 126gcatagatac
cgtctcttga tctgaa 2612720DNAArtificial SequenceSynthesized primer
127aggtggagga agcagtgaga 2012820DNAArtificial SequenceSynthesized
primer 128gcttggggtt gtgaaagaaa 2012925DNAArtificial
SequenceSynthesized primer 129aatccaccaa agctcacgcg tggaa
2513025DNAArtificial SequenceSynthesized primer 130tgatgtgtct
ctcggtcaag ttcaa 2513123DNAArtificial SequenceSynthesized primer
131aagtgtggaa ctctctggaa ctg 2313223DNAArtificial
SequenceSynthesized primer 132gggttatctt ggttggcttt atg
2313320DNAArtificial SequenceSynthesized primer 133tggagactgc
tctggaaggt 2013420DNAArtificial SequenceSynthesized primer
134tgctgctgtc ctcaaatacg 2013520DNAArtificial SequenceSynthesized
primer 135ccaaaaagac ctcgttcagc 2013620DNAArtificial
SequenceSynthesized primer 136atgtctccgg atcgtttcac
2013728DNAArtificial SequenceSynthesized primer 137ccagcacctt
cagcttcagg tccttgat 2813828DNAArtificial SequenceSynthesized primer
138tgccggaagt tgagaggaaa tccaagat 2813921DNAArtificial
SequenceSynthesized primer 139accaagggct cagagcatgc a
2114023DNAArtificial SequenceSynthesized primer 140tggctttcag
gagagtatct ttg 2314122DNAArtificial SequenceSynthesized primer
141ccgcaaggga aagatgaaag ac 2214220DNAArtificial
SequenceSynthesized primer 142tcgtttggtt tcggggtttc
2014324DNAArtificial SequenceSynthesized primer 143gccagcctct
cctgatttta gtgt 2414424DNAArtificial SequenceSynthesized primer
144gggaacacaa aagacctctt ctgg 2414521DNAArtificial
SequenceSynthesized primer 145tcccgcagcc gagccgcggg g
2114627DNAArtificial SequenceSynthesized primer 146tcttccaact
gcctctctgg ccctccg 2714724DNAArtificial SequenceSynthesized primer
147catttctcca cctcactgaa actg 2414823DNAArtificial
SequenceSynthesized primer 148aatgtgttag tgcatgcaca cac
2314920DNAArtificial SequenceSynthesized primer 149aagggtggag
gtgtcttggt 2015022DNAArtificial SequenceSynthesized primer
150gcttctttac actcctgcta aa 2215123DNAArtificial
SequenceSynthesized primer 151ctaaggatgg tggagaacct
tca 2315222DNAArtificial SequenceSynthesized primer 152cgcgcttaat
agtgtccatc ag 2215322DNAArtificial SequenceSynthesized primer
153atccaattgt gccaagcact aa 2215425DNAArtificial
SequenceSynthesized primer 154ccaccatttt gatagtttcc atagg
2515522DNAArtificial SequenceSynthesized primer 155ctcgacaact
tcccagataa cc 2215625DNAArtificial SequenceSynthesized primer
156ggtacaggtt gcgatagatg atagc 2515720DNAArtificial
SequenceSynthesized primer 157gccccaattc gcgccaagga
2015820DNAArtificial SequenceSynthesized primer 158aggttggcgg
cagttctggg 2015920DNAArtificial SequenceSynthesized primer
159ttcttccatg tccgggatag 2016020DNAArtificial SequenceSynthesized
primer 160ccccactctt ctcgaatcag 2016125DNAArtificial
SequenceSynthesized primer 161gctggacgtg atcttactga ttacc
2516224DNAArtificial SequenceSynthesized primer 162gtagcagagc
ttctccttga tgtc 24
* * * * *
References