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Somatotropic (GH) effects on methionine metabolism and aging
International Symposium on Geroscience2016
Acknowledgements
UNDSharlene RakoczyVanessa ArmstrongDeb RaasakkaNicole RaasakkaJoy RojanathammaneeJoe Wonderlich
SIUAndrzej Bartke
USDA HNRCEric Uthus
University of GlasgowPeter AdamsJohn ColeNeil RobertsonMohammad Iqbal Rather
Support: Glenn Foundation for Medical ResearchNIH National Institute on AgingEllison Medical Foundation
Take home message: Reduced somatotropicsignaling allows for metabolic reprogramming –
shifting resources away from growth & proliferationtowards stress resistance & cytoprotection.
Outcome of reprogramming: A more stableepigenome that in turn, results in extendedhealth and life spans.
Endocrine Lifespan Plasma Stress Insulin Genotype effect extension GH resistancesensitivity Reference Amesdwarf GHdeficient 49-68% Brown-Borgetal.,1996Prop1df +PRL&TSH
deficientAmesdwarf ““ +12% ND Bartkeetal.,2001+calorierestrictionSnelldwarf GHdeficient 42-50% Flurkeyetal.,2001Pit1dw +PRL&TSH
deficientLittle GHdeficient 23-25% Flurkeyetal.,2001GHRHRlitGHRH-/- GHdeficient 43-51% Sunetal.,2013GHR/BP-/- GHresistant 38-55% Coschiganoetal.,2000(GHRKO) GHRKO ““ noincrease ND Bonkowskietal.,2006+calorierestriction FGF21Tg GHresistant 36% ND Zhangetal.,2012IGF1R+/- partialGH 33%*+ ND Holzenbergeretal.,2003
resistance
IGF1R+/- partialGH 5%* ND Bokovetal.,2011 resistance
*
*
**
**some studies have been repeated on different background strains with same* or different results**
Phenotypic Characteristics of GH/IGF Mutant Mice
GHRH SS
liver
AnteriorPituitaryGH (+)
Target cells
IGF-1 (+)IGF-1 (+)
IGFBP (-)
IGFBPproteases (-)
Somatotropic axis mutants
IGF-1 (+)
LittleGHRHKO
Ames, Snell
GHRKO
IGF1R+/-
Klotho tgMidiPAPP-A Reduced IGF-1 levels/signaling
Lifespan
49-68%
Ames Mice Prop-
1 gene mutation
GH, prolactin,
TSH deficient
Insulin sensitivity
Stress resistance
Growth Hormone Mutant Mice
0 5 10 15 20 25 30 35 40 45 500
10
20
30
40
50
60
70
80
90
100 Normal
Dwarf
Months
Perc
en
t su
rviv
al
wild typedwarf
Kidney aging
Immune system aging
Cataract development
Collagenaging
Neuromuscular dysfunction
Joint cartilage & osteoarthrosis
Learning & memory loss
Fatal disease
Cancer incidence
Glucose homeostasis disruption
GH deficient mice have longer healthspans
Guivere-Aguirre et al, 2011
GH receptor deficiency
****
GH & IGF-1 in Humans*Congenital IGF-1 deficiency confers protection against cancer, diabetes (Steuerman et al., 2011; Guivere-Aguirre et al., 2011)
Intrachromasomal interactions among IGF1R, IRS2 & UCP2 associated with longevity (Barbieri et al., 2012)
*Insulin/IGF1 pathway mutations associated with longevity (van Heemst et al., 2005; Pawlikowska et al., 2009; Willcox et al., 2008; Flachsbart et al., 2009; Sorensen et al., 2010, 2012; Milman et al., 2014)
IGF1R gene mutations associated with longevity (Bonafe et al., 2005; Suh et al., 2008)
*Impaired IGF1R signaling in cells expressing longevity–associated human IGFR1 alleles (Tazearslan et al., 2011)
Delays in aging/age-related disease & increased longevity may result from overall enhanced stress resistance due to decreased
signaling via GH/IGF1 pathways
Dwarfism - Prop-1 gene mutation
GH, prolactin & TSH deficient
Increased life span (49-68%)
Enhanced insulin sensitivity
Enhanced stress resistance
Growth Hormone Mutant Mice
Overexpresses bovine GH (PEPCK)
Very high plasma [GH]
Decreased life span (50%)
Hyperinsulinemic, insulin resistant
Decreased stress resistance
Ames dwarf GH transgenic
GH transgenic mice have longer healthspans
• Incidence of cancer is reduced
• Fatal disease develops later in life
• Youthful cognitive function (learning & memory) is maintained
• Immune system aging delayed
• Cataract development delayed
• Kidney GBM thickening delayed
• Joint cartilage & osteoarthrosis delayed
• Reproductive senescence accelerated
Mechanisms of Cellular Stress Resistance/Defense
Scavenging systems (antioxidative defense)
tissue specific systems
Detoxification (GSTs)
Heat shock proteins
Metal chelators Apoptosis
Repair systems (DNA, protein)
Your ‘pet’mechanism
**GH status impacts many of these systems**
*Total tissue GSH pool > in Ames dwarf mice*
One mechanism underlying elevated GSH levels = altered GSH metabolic pathway
Glutathione Metabolism
Cysteine
-Glutamylcysteine
Glutathione
GSSG
Glutamylcysteine ligase
GSH synthase
GPX GR
GGT
exogenous &endogenous cpds
GSH-conjugatedcpds (detoxified)
O2_. ROS (H2O2)
ROH (H2O)
GSH S-transferase
-glutamyl transpeptidase
*GH
Liver GST/4HNE Activity
WT saline dwarf saline dwarf GH0
200
400
600
nm
ol/m
in*m
g
****GSTA1
Glutathione (GSH)metabolism of toxins/carcinogens, antioxidant,DNA synthesis & repair, protein synthesis
Methionine (MET)Essential amino acid
-GSH synthesis-maintenance of intracellular GSH pool-protein synthesis
S-adenosylmethionine (SAM)-methyl donor
Why is methionine important in aging?
• Calorie (CR), protein (PR) & methionine restriction (MR) extend lifespan in rodents & other species
• CR & MR are associated with attenuation of age-related disease in mammals
• Dietary reduction in calories or protein consequently reduces MET
• Methyl deficient diets induce liver injury /genetic defects in MET pathway associated with age-related pathologies
• Methionine, homocysteine, folate, choline & betaine –severe deficiencies & overexpression associated with disease
• CR, PR & MR associated with an in resistance to cellular stressors
• MET metabolism is central to cellular methylation & redox buffering processes
Methionine metabolism is enhanced in GH deficiency
Methionine
SAM
SAH
Homocysteine
Cystathionine
Cysteine
-Glutamylcysteine
Glutathione
GSSG
MAT
GNMT
SAH hydrolase
CBS
CTH
GCL
GSH synthase
GPX GR
GGT
exogenous &endogenous cpds
GSH-conjugatedcpds (detoxified)
GST
MET synthase
(Folate pathway)
Dwarf
+GH
(mRNA, protein, activity)
transsulfuration
transmethylation
Normally: When MET is abundant – transsulfuration is favoredWhen MET is low – transmethylation is favored
Methyl-moiety & carbon chain are lost much faster in dwarf mice transmethylation &transsulfuration are bothincreased
SAM & SAH are altered in Ames mice
SAM is universal methyl donor-95% of all SAM is used for methylation (wide variety of molecules)
-85% of all transmethylation reactions occur in liver*
Dwarf mouse exhibits atypical methionine metabolism
3H 35S0
10
20
30
40
50
60
dwarf
wild type
pC
i/nm
ol S
AM
***
*p<0.05**p<0.005
Methionine Flux
3 month 12 month 24 month
0
1
2
3
4
5
Interaction p= 0.0011Genotype p<0.0001Age p<0.0001
****Dwarf
WT
DNM T3a mRNA
Re
lativ
e E
xp
res
sio
n
3 month 12 month 24 month
0.0
0.5
1.0
1.5
2.0
2.5
Interaction p= 0.0350Genotype p<0.0001Age p=0.0002
****DNM T1 mRNA
Dwarf
WT
Re
lativ
e E
xp
res
sio
n
Liver DNA methyltransferase expression & activity
Dnmt1 – maintenanceDnmt3a, 3b - de novo
3 months
0.00
0.05
0.10
0.15
***
DN
MT
1
Me
an
Op
tic
al D
en
sit
y
0.00
0.01
0.02
0.03
0.04
0.05
***
DN
MT
3a
Mea
n O
pti
ca
l D
en
sit
y
12 months
0.00
0.05
0.10
0.15
*
0.00
0.02
0.04
0.06
0.08
0.10
24 months
0.00
0.05
0.10
0.15
0.20
0.25
**
0.00
0.05
0.10
0.15
0.20
*
Protein expression
0.0
0.5
1.0
1.5
12 months
0.0
0.5
1.0
1.5 * DwarfWT
3 months
rela
tiv
e D
NM
T a
cti
vit
y
0.0
0.5
1.0
1.5
*
24 months
DNMT ActivityD
nm
t1D
nm
t3a
DNMT1
WT saline Dwarf saline Dwarf GH
0
1
2
3
Re
lativ
e E
xp
res
sio
n
**
WT Saline Dwarf Saline Dwarf GH
0.00
0.05
0.10
0.15
** *
Me
an
Op
tic
al D
en
sity
DNMT3a
WT saline Dwarf saline Dwarf GH
0
1
2
3
Re
lativ
e E
xp
res
sio
n
WT Saline Dwarf Saline Dwarf GH
0.00
0.05
0.10
0.15
*
GH treatment Dnmt1 & Dnmt3a protein in dwarf mice
mRNA
protein
GH also treatment impacts Dnmt1 & Dnmt3a protein in vitro.
0.16% 0.43% 1.3%0.0
0.5
1.0
1.5
2.0
2.5
Rela
tive E
xp
ressio
n
DNMT1
Wild type
Dwarf****
Interaction p=0.0135Diet p=0.0054Genotype p<0.0001
0.16% 0.43% 1.3%0
1
2
3
4
Rela
tive E
xp
ressio
n
DNMT3A
Wild type
Dwarf
Interaction p=0.0436Diet p=0.0582Genotype p<0.0001
*
****
0.16% 0.43% 1.3%0
1
2
3
4
Rela
tive E
xp
ressio
n
DNMT3B
Wild type
Dwarf
Interaction p=0.0567Diet p=0.0651Genotype p<0.0001
**
****
DNA methyltransferase gene expression is elevated in dwarf mice on diets with varying levels of MET
• Dwarf mice exhibit atypical methionine metabolism & GHappears to regulate enzymes in pathway
• Flux of methionine is 2-3X greater in dwarf compared to wild type mice thus, transmethylation & transsulfurationare increased
• Liver DNA methyltransferases (Dnmt1, 3a) are increased in GH deficiency & modulated by GH (mRNA, protein, activity)
**Based on this evidence, we predicted that the dwarf epigenome may be more stable than wild type mice as they age
Metabolic characteristics that may contributeto altered epigenome in long-living mice
DNA Methylation in Dwarf Mice
Liver DNA methylome of wild type and Ames dwarf mice
2 month wild type (WTY) 2 month dwarf (DY)22 month wild type (WTO) 22 month dwarf (DO)
4 male replicates; 15x coverage; 1440Gbp sequence
0%
20%
40%
60%
80%
100%
DY DO NY NO
Glo
bal
% m
eth
tyla
ted
Cp
Gs
WTY
WTO
DY
DO
Chr1195Mb
-indicates that there are no largescale changes in the epigenome
Peter Adams
-150
-50
50
150
250
-200 -100 0 100 200
PC
2
PC1
WTY WTO DY DO
0
10000
20000
30000
40000Total
Hypomethylated
Hypermethylated
Dwarf Normal
Ch
ange
in m
eth
ylat
edC
pG
s w
ith
age
Differentially methylated regions between young and old wild type (>=10%)
DY
DO
WTY
WTO
Liver DNA methylome of Ames dwarf mice is more STABLE compared to wild type mice
Peter Adams
0
1000
2000
3000
4000
-91
%
-71
%
-51
%
-31
%
-11
%
9%
29
%
49
%
69
%
Dwarf
NormalNormal
Dwarf
Nu
mb
er o
f D
MR
s
Difference in methylation (O-Y)
DY
DO
WTY
WTO
Liver DNA methylome of Ames dwarf mice is BUFFEREDcompared to wild type mice
Peter Adams
DMRs hypomethylated with age in both wild type and Ames mice
30%
40%
50%
60%
70%
80%
Mea
n %
met
h p
er s
amp
le
WTY WTO DY DO
Hypomethylation of aDMRs - genomic features aresimilar between genotypes but WT mice harbor significantly greater numbers of hypomethylated genes & enhancers (little overlap on CpG islands).
Higher level of methylation in young dwarf serves tobuffer against age-associated hypomethylation.
Ames mice respond differently to altered dietary MET compared to wild type mice (median-log-rank; maximal–Fisher test)
Median and maximal lifespans
0.16% MET Dwarf = WT Lifespan extension in wild type mice
0.43% & 1.3% MET Dwarf > WT
Lifespan Study
Several studies have shown that MET restriction extends lifespan
0 250 500 750 1000 1250 1500
0
20
40
60
80
100
WT 0.16%
Dwarf 0.16%
Days
Pe
rce
nt
su
rviv
al
Median p=.135890th % p=.4982
0 250 500 750 1000 1250 1500
0
20
40
60
80
100
Days
Perc
en
t s
urv
iva
l WT 0.43%
Dwarf 0.43%
Median p<.000190th % p=.0003
0 250 500 750 1000 1250 1500
0
20
40
60
80
100
Days
WT 1.3%
Dwarf 1.3%
Pe
rce
nt
su
rviv
al
Median p<.000190th % p=.0005
0 250 500 750 1000 1250 1500
0
20
40
60
80
100
Days
Pe
rce
nt s
urv
iva
l
GHRKO 0.16%
GHRKO 0.43%
GHRKO 1.3%
0 250 500 750 1000 1250 1500
0
20
40
60
80
100
Days
Pe
rce
nt
su
rviv
al
GH Tg 0.16%
GH Tg 0.43%
GH Tg 1.3%
Median
0.16 vs 0.43 p<.0001
0.16 vs 1.3 p<.0001
90th %
0.16 vs 0.43 p=.0056
0.16 vs 1.3 p=.0060
0 250 500 750 1000 1250 15000
20
40
60
80
100
Days
Pe
rce
nt s
urv
iva
l
Dwarf WT 0.16%
Dwarf WT 0.43%
Dwarf WT 1.3%
Median0.16 vs 0.43 p<.0001
0.16 vs 1.3 p<.0001
90th %0.16 vs 0.43 p=.0164
0.16 vs 1.3 p=.0012
0 250 500 750 1000 1250 15000
20
40
60
80
100
Days
Pe
rce
nt
su
rviv
al
GHRKO WT 0.16%GHRKO WT 0.43%
GHRKO WT 1.3%
Median0.16 vs 0.43 p<.00010.16 vs 1.3 p<.0001
90th %
0.16 vs 0.43 p=.00120.16 vs 1.3 p=.0013
0 250 500 750 1000 1250 15000
20
40
60
80
100
Days
Pe
rce
nt
su
rviv
al
GH Tg WT 0.16%
GH Tg WT 0.43%
GH Tg WT 1.3%
Median
0.16 vs 0.43 p<.00010.16 vs 1.3 p=.0004
90th %0.16 vs 0.43 p=.0003
0.16 vs 1.3 p=.0295
0 250 500 750 1000 1250 15000
20
40
60
80
100
Days
Pe
rce
nt s
urv
iva
l
Dwarf 0.16%
Dwarf 0.43%
Dwarf 1.3%
Reduced somatotropic signaling
Enhanced methionine metabolism
methyltransferase activity maintenance of DNA methylation pattern
epigenetic stability
cysteine availability thiol compounds
GSH metallothionein
antioxidative defense, potent antioxidant,maintains GSH for redox selenoprotein R metal chelator,
& enhances detoxification (MsrB) protein repair, **Dnmt1-regulated**recycling of MET
(normal chow, MR, ME)
Conclusions
• GH status impacts MET metabolism & integrated pathways(regardless of dietary MET intake)
• GH signaling is necessary to discriminate levels of dietary MET
• The DNA methylome of GH-deficient mice appears to be:more STABLE than wild type micebetter BUFFERED against changes than wild type micebetter CONTROLLED (more uniform between mice) than wild type
• GH causes a shift in resources - In presence of GH increased demand for amino acids for growth In absence of GH shift in resources towards defense mechanisms
GH is promoted as an ‘anti-aging’ factor yet it suppresses defense mechanisms, impacts nutrient sensing, alters DNA methylation & accelerates age-related disease processes.
Thank you!