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!