Nir Barzilai M.D.Director: Institute for Aging Research

Departments of Medicine

and Molecular Genetics

Cambridge, September 2003Cambridge, September 2003

Longevity-biology of caloric restrictionXiao-Hui Ma M.DXiao-man Yang M.D.Radhika Muzumdar M.D.Bing-Qian Liu M.DIlan Gabriely M.D.Patricia Vuguin M.D.

Thank you longevity!!!

Longevity-genes Gil Atzmon Ph.D. Bill Greiner R.N. Magda Gabriely M.D.Debra Davidson M.Sc.Clyde Schechter M.D.Richard Lipton M.D.Ms. Marion MazeMs. Linda Radner

Other CollaboratorsAlan Shuldiner M.D. (UMD)Braxton Mitchell Ph. D. (UMD) Hassy Cohen M.D. (UCLA)Tom Perls M.D. (BU)Gad Rennert M.D., Mp.H.(Technion)Amiela Globerson Ph.D. (Ben Gurion)

Norman Fleischer M.D (Einstein)Philip Scherer Ph.D. (Einstein)Becky Simmons M.D. (Penn)

Program project Jill Crandall M.D.Meredith Hawkins M.D.Harry Shamoon M.D. Luciano Rossetti M.D.Michael Brownlee M.D.Silvana Obici M.D.

65 millions year 50 years

65 millions year 50 years

Antagonistic Pleiotropy

The metabolic syndrome of aging:

Obesity/abdominal obesityType 2 diabetes mellitusDyslipidemia

ThrombosisEndothelial dysfunctionInflammationHypertension

Insulin resistance

ASCVDCancer

PAI-1

IL-1, IL-6, CRPAGT Low HDL, ‘small’ LDL

High insulin

Nutritional (transcription) regulation of peptides Leptin

2 am

8 am 8 amGlucose-insulin/FFA/AA

How do nutrients induce biological effects?

Leptin fails to regulate body fat distribution,insulin action, and endocrine functions with aging

Barzilai JCI 1997,Rossetti & Barzilai Nature 1999, Gabriely & Barzilai Diabetes 2002,Xma & Barzilai J Gerontol 2002.

FFA

How is leptin expression regulated? Hexosamine Biosynthetic Pathway

Glucose

Glucose-6-P

Glc-1-P

UDP- Glc

Glycogen

F -6-P

Triose -P

Glycolysis

GlcN-6-P

UDPGlcNAc

GFAT

1%FFA

Glucosamine

GlycosylationSp1

NFkBPGC1

--3-6%

AA (Glutamine)

How can a pathway serve as ‘nutrient sensing’?

• Should serve as a switch “on” or “off”.

• Should be a small pathway that can be amplified by several folds.

• Should be linked to a biological effect (transcription).

• Should exist in every organ, although may be distinct.

• Should apply to all models of CR

Although old CR animals are metabolically similar to young ratsthey have increased levels of muscle hexosamines (p<0.01)

*

Old CR

Muscle UDPGlcNAc/UDPGlc

0

1

2

3

Young

**

Old AL

Is the HPB activated with aging?

Insulin glucose isotopes infusion

Blood

Sample

Does the HBP effect the muscle function of old CR rats?

Insulin-3 mU/kg/minGlcN-7 µM/kg/min/or Saline

3 mo (n=5) and 21 mo (n=5) CR S-D rats

Time (min.)

0

5

10

15

20

25

30

30 50 70 90 110150190210230250270290

Young Old

Rd (mg/kg/min)

* * * *

* * * *

* * * *

P<0.001

Young/old CR saline control

UDP-GlcNAc 20.8 nmol/g muscle

UDP-GlcNAc 17.7 nmol/g muscle

Excess nutrients can induce peripheral insulin resistance through the HBP!

(and more so in aging)

Does the HBP affect adipose tissue function of old CR rats?

PAI-1 expression(Real-time PCR)

1

3.22.2

12.5

0

2

4

6

8

10

12

14

Saline GlcN

YoungOld

Variety of (harmful?) fat-derived peptides are over expressed through the HBP.

Is the transcription of PAI-1 by the HBP associated with increase in plasma levels?

0

5

10

15

20

25

30

+ - + -

Young Old CRGlc

*

**

PAI-1 activity U/ml

Artheriosclerosis. 160: 117. 2002.

Is increased availability of FFA effect PAI-1 transcription in humans adipose tissue?

Ge

ne

ex

pre

ss

ion

lev

el

(re

lati

ve t

o b

asel

ine)

Liposyn - +

PAI-1ß-actin

0

10

20

30

Hawkins et al

Similar effects have been induced by hyperglycemia, hyperinsulinemia, and increased levels of amino acids in humans and rodents

Which organ is most important toexert the biological effects of nutrient excess on longevity?

Does VF account for the effects of caloric restriction on insulin action in aging rats?

Body Weight (g)0

100

200

300

400

500

Young Old SC- Old VF- Old CR

Diabetes; 51:2951, 2002

0

10

20

30

Rd (mg/kg LBM/min)

*

Is the removal of VF sufficient to restore insulin action?

*P<0.01

Young SC- VF- CR

Diabetes; 51:2951,2002

Does ‘knock out’ of VF prevents diabetes in Zucker diabetic rats?

0

20

40

60

80

100

120

2 mo 3 mo 4 mo 5 mo 6 mo

ZDVF-ZDVF+

~40%

~80%%DM

Diabetes; 51:2951,2002

02468

10121416

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PAI-1

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Nutrients, nutrient sensing, and induction of fat-derived peptides

SalineGlucoseInsulinGlcN+In

Resistin

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60

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SalineGlucoseInsulinGlcN+In

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0

2

4

6

8

10

12

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SalineGlucoseInsulinGlcN+In

Angiotensinogen

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IL-6IL-1ResistinSAA

PAI-1AGT

How do nutrients induce biological effects?

FAT+Nutritional (transcription) regulation of peptides

Leptin 2 am

8 am 8 amGlucose/insulin/FFA

Clinical trials are standardizes by fasting levels, and areUnder estimating daily transcription of peptides!

But

You never know the whole truth!

You always understand the results of your

experiment!

98

95

92

100

8.6

8.8

9.0

9.2

9.4

9.6

9.8

60 65 70 75 80 85 90 95 100<

Age

HD

L P

arti

cle

Siz

e (n

m)

LD

L P

arti

cle

Siz

e (n

m)

ControlOffspring

Probands

20.4

60 65 70 75 80 85 90 95 100<

Age

20.6

20.8

21.0

21.2

21.4

21.6

Lipoprotein particle sizes with age

JAMA (October 03 )

Heritabilty of lipoprotein size 0.4-0.7!

Is lipoprotein sizes associated with HTN or CVD?(in offspring of centenarians)

20

30

40

50

60

% large HDL size

Healthy (n=209HTN (n=64)CVD (n=20)

*

*P<0.003

20

30

40

50

60

70

% large LDL size

*

JAMA (October 03 )

TESSSESIQSFLQSMITExon 14

ATC

GTC

= Isoleucine

= Valine

A

A

Homozygous

II

OR

Heterozygous

A

G

IV

OR

Homozygous

G

G

VV

Cholestryl Ester Transfer Protein (CETP)

0

10

20

30

40

50

60

70

II IV VV

Per

cen

tag

e in

po

pu

lati

on

Control

Offspring

Probands

* ***

CETP I 405 V frequency in families with exceptional longevity

CETP VV genotype attributes to 18% of longevity

JAMA (October 03 )

IS CETP genotype associated with its plasma levels?

1

1.2

1.4

1.6

1.8

2

2.2

2.4

CETP levels (ug/ml)

I/II/VV/V

*P<0.02

*

JAMA (October 2003 )

Is lipoprotein sizes a risk for MS ?(in offspring and spouse of offspring)

JAMA (2003, 290;15;2030-40 )

% large LDL size % large HDL size

Healthy (n=221)MS (n=47)

30

35

40

45

50

55

60

30

35

40

45

50

55

60

65

70

***P<0.001

******

0

10

20

30

40

50

60

70

CC CA AA

Per

cen

tage

in p

opu

lati

on

Control

Offspring

Probands

APOC3 C(-641)A frequency in families with exceptional longevity

******

***P<0.001

0

20

40

60

80

100

Ali

ve

60 80 100 120 140

Age

Rate limiting steps for humans longevity

cancer

CVD

CR

Telomeres

Mitochondria

Barzilai lab

Revisiting the role of fat mass in the life extension induced by caloric restriction

• J. Gerontol Barzilai & Gupta; 1999, 54:B89

…This hypothesis should not be revisited

...This is not a novel hypothesis

Mounting evidence supports the notion that fat mass is inert and plays no role, as compared with nutrients per se in CR.

The role of fat mass as causative of age-related diseases leading to morbidity and mortality is well accepted. Type 2 DM is a striking example.

Insulin

glucose

isotopes

infusion

Blo

od

Sam

ple

Rueben AndresFault!

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Nutrients, nutrient sensing, and induction of fat-derived peptides

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0

2

4

6

8

10

12

SC VF

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Angiotensinogen

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Probability of survival in caloric restricted animals

Months

Su

rviv

al

100%Decrease in all causes

of deaths

Human longevity genesWe hypothesize that to get to age 100 (1/10,000) one needs longevity assurance genes.

Offspring inherit longevity traits from their parents, and these may be a platform for longevity-associated traits.

We have collected over 1000 samples! (At their homes)

Our population of (Ashkenazi) Jews is genetically homogenous, increasing the likelihood of genetic discoveries.

Funding by AECOM the National Institute of Aging and the Ellison medical Foundation

Lipoprotein sizes and Cognitive function (by MMSE) in centenarians

0

20

40

60

80

100

% large LDL size

MMSE>25MMSE<25

20

20.3

20.6

20.9

21.2

21.5

LDL size (nm)

Lipoprotein sizes and I405V genotype

20

30

40

50

60

% large HDL size

I/II/VV/V

*

*P<0.001 vs. I/I

20

30

40

50

60

70

% large LDL size

*

JAMA (In Press )

Does leptin resistance effect insulin action with aging? % suppression of hepatic glucose production

-100

-80

-60

-40

-20

0

Young Old CR

Leptin vs. pair-fed

*

Gabriely et al Diabetes 51:1016, 2002

Leptin resistance may lead to alteration in body fat-distribution and insulin resistance!

Mitochondrial Overproduction of Superoxide Activates Four Mitochondrial Overproduction of Superoxide Activates Four Major Pathways of Hyperglycemic Damage By Inhibiting Major Pathways of Hyperglycemic Damage By Inhibiting

GAPDHGAPDH

Brownlee M. Nature. 2001;414:813-820.

NADPH

GFAT

Gln Glu

NADH

Sorbitol

NADP+ NAD+

NAD+

NADH

Glucosamine-6-P UDP-GlcNAc

Glucose

Glucose-6-P

GAPDH

Fructose-6-P

1,3-Diphosphoglycerate

Glyceraldehyde-3-P

Fructose

NADH NAD+

-Glycerol-P DAG PKC

Methylglyoxal AGEs

DHAP

O2–•

Hexosamine pathway

Protein kinase C pathway

AGE pathway

Polyol pathway

Does the HBP affect adipose tissue function of old CR rats?

1

3.22.3

11

0

2

4

6

8

10

12

Saline GlcN

YoungOld

Leptin expression(Real-time PCR)

Does leptin fail to regulate VF with aging?

-60

-50

-40

-30

-20

-10

0

Young Old CR

% decrease Leptin vs. pair-fed

*

Barzilai JCI 1997,Rossetti & Barzilai Nature 1999 Gabriely & Barzilai Diabetes 2002,Xma & Barzilai J Gerontol 2002.

Leptin resistance may lead to alteration in body fat-distribution and to insulin resistance!

Out off approximately 7,000 full-length sequences and approximately 1,000 EST clusters

2570 were expressed

200 were up/down- regulated in each chip

Atzmon et al Horm Metab Res. 2002 34:622

In VF In SCInsulin-induced growth-respons protein (CL-6) 5.9 -Beta 3-adrenergic receptor 5 -Phosphoenolpyruvate carboxykinase (GTP) PEPCK 4.3 -PPAR-gamma 4.1 -Hormone sensitive lipase 3.5 -Insulin-like growth factor I 3.2 -Fatty acid transporter 3.2 -Thioesterase II - 156Mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase - 21.1Low molecular weight fatty acid binding protein - 10.1GLUT1 = glucose transporter 1 - 9.8Lipopolysaccharide binding protein - 9.7Lysosomal acid lipase = intracellular hydrolase - 4.6Fatty acid synthase - 3.9Type II cAMP-dependent protein kinase regulatory subunit 7.4 -Adipocyte hormone-sensitive cyclic AMP phosphodiesterase 5.7 -Potential-sensitive polyspecific organic cation transporter 5.6 -Retinol-binding protein (RBP) gene, exon 5 5.2 -Steroidogenic acute regulatory protein 5 -Growth hormone receptor 5 -

Chaperonin 60 (Hsp60) and chaperonin 10 (CPN10) genes, nuclear genes encoding mitochondrial proteins

4.8 -

Aquaporin 7 4.3 -Angiotensinogen 4.5 -Glutathione-dependent dehydroascorbate reductase 4 -MHC class II antigen RT1.B-1 beta-chain 3.6 -Tricarboxylate carrier 3.6 -Thyroid stimulating hormone receptor 3.6 -Phosphodiesterase I 3.5 -Water channel aquaporin 3 (AQP3) - 20.8Carbonic anhydrase II - 11.8Wistar-Kyoto (Heidelberg) angiotensin converting enzyme - 7.4GST - 6.1Na-K-Cl cotransporter (Nkcc1) - 5.7Alpha-2-u globulin - 5.3Glutathione S-transferase Yc1 subunit - 4.7Wistar transforming growth factor beta-3 - 4.7Liver glutathione S-transferase Yc subunit - 4.4Polymeric immunoglobulin receptor - 4Alkaline phosphatase - 3.9

Cellular metabolism and other

Gene PathwaysMultiple increase

Glucose homeostasis Insulin action and lipid metabolism

Atzmon et al Horm Metab Res. 2002 34:622