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Cardiac Specific Overexpression of Insulin-Like Growth Factor-1 (IGF-1)

Attenuates Aging-Associated Cardiac Diastolic Contractile Dysfunction and

Protein Damage

Qun Li1, Shan Wu1, Shi-Yan Li1, Faye L. Lopez1, Min Du2, Jan Kajstura3, Piero Anversa3

and Jun Ren1

1Division of Pharmaceutical Sciences & Center for Cardiovascular Research and

Alternative Medicine; 2Department of Animal Sciences, Laramie, WY 82071; 3Department

of Medicine, New York Medical College, Valhalla, NY 10595

Running head: IGF-1 overexpression attenuates cardiac aging

Correspondence to:

Dr. Jun Ren

Division of Pharmaceutical Science & Center for Cardiovascular Research and Alternative

Medicine, University of Wyoming, Box 3375, Laramie, WY 82071-3375.

Tel: (307)-766-6131; Fax: (307)-766-2953

E-mail: jren@uwyo.edu

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Articles in PresS. Am J Physiol Heart Circ Physiol (November 3, 2006). doi:10.1152/ajpheart.01036.2006

Copyright © 2006 by the American Physiological Society.

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ABSTRACT

Aging is associated with hepatic growth hormone resistance resulting in a fall in serum insulin-

like growth factor-1 (IGF-1) level. However, whether loss of IGF-1 contributes to cardiac aging

is unclear. This study was designed to examine the effect of cardiac overexpression of IGF-1 on

cardiomyocyte contractile function in young (3 mo) and old (26-28 mo) mice. Cardiomyocyte

contractile function was evaluated including peak shortening (PS), time-to-90% PS, time-to-90%

relengthening (TR90) and maximal velocity of shortening/relengthening (± dL/dt). Levels of

advanced glycation endproduct (AGE), protein carbonyl, sarco(endo)plasmic reticulum Ca2+-

ATPase (SERCA2a), phospholamban (PLB) and Na+-Ca2+ exchanger (NCX) were assessed by

Western blot. SERCA activity was measured by 45Ca2+ uptake. Aging induced a decline in

plasma IGF-1 levels. Aged cells exhibited depressed ± dL/dt, prolonged TR90 and a steeper PS

decline in response to increasing stimulus frequency compared with young myocytes. IGF-1

transgene alleviated aging-induced loss in plasma IGF-1 and aging-induced mechanical defects

with little effect in young mice. The beneficial effect of IGF-1 transgene on aging-associated

cardiomyocyte contractile dysfunction was somewhat mimicked by short-term in vitro treatment

of recombinant IGF-1 (500 nM). AGE and protein carbonyl levels were higher in aged mice

which were not affected by IGF-1. Expression of SERCA2a (but not NCX and PLB) and

SERCA activity were reduced with aging, which was ablated by the IGF-1 transgene.

Collectively, our data suggest beneficial role of IGF-1 in aging-induced cardiac contractile

dysfunction, possibly related to improved Ca2+ uptake.

Key words: IGF-1, cardiomyocytes, aging, contractile function, Ca2+ regulatory protein.

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INTRODUCTION

Cardiac aging is a continuous and irreversible biological process contributing to the high

morbidity and mortality in the elderly (4; 10; 11). Several theories have been made for cardiac

aging including prolonged action potential duration, myosin heavy chain isozyme switch,

impaired intracellular Ca2+ homeostasis, altered membrane structure and permeability and

accumulation of reactive oxygen species (ROS), which may lead to abnormal cardiac contractile

function (8-10). In addition, aging induces reduction in growth hormone (GH) and subsequent

insulin-like growth factor-1 (IGF-1) deficiency, both of which essential for the maintenance of

normal cardiac structure and function. GH/IGF-1 deficiency has been reported to be associated

with altered body composition, cytokine and neuroendocrine activation, cardiac atrophy and

impaired cardiac function. The risk of cardiovascular disease is increased in subjects with IGF-1

deficiency. IGF-1, the mediator of many of GH-associated effects in peripheral tissues, improves

myocardial function in the setting of both healthy and failing hearts (22). The therapeutic

potential of IGF-1 has been implicated in cardiac disorders related, but not limited, to heart

failure, myocardial infarction and diabetes (22). Nonetheless, the role of IGF-1 in cardiac aging

remains largely obscure. Therefore, the present study was designed to determine if cardiac-

specific overexpression of IGF-1 alleviates aging-associated cardiomyocyte contractile function

and affects expression/function of key Ca2+ regulatory proteins.

MATERIALS AND METHODS

Experimental animals

All animal procedures used in this study were approved by the Animal Care and Use

Committees at the University of North Dakota (Grand Forks, ND) and the University of

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Wyoming (Laramie, WY). Male FVB and IGF-1 heterozygous transgenic mice at young (3

month-old) and old (26-28 month-old) ages were used. The pigmentation of fur color was used

as a marker for heterozygous IGF-1 (light brown) or wild-type FVB (white) mouse identification

as described (20). All animals were kept in our institutional animal facility at the University of

Wyoming with free access to standard lab chow and tap water. At the time of sacrifice, blood

glucose and plasma IGF-1 levels were measured using a glucose monitor (Accu-ChekII, model

792, Boehringer Mannheim Diagnostics, Indianapolis, IN, USA) and an ELISA commercial kit

from Diagnostic System Laboratory (Webster, TX, USA), respectively.

Isolation of mouse ventricular myocytes

Hearts were rapidly removed from anesthetized mice and mounted onto a temperature-

controlled (37°C) Langendorff system. After perfusing with a modified Tyrode solution (Ca2+ free)

for 2 min, the heart was digested for about 10 min with 0.9 mg/ml collagenase D (Boehringer

Mannheim Biochemicals) in the modified Tyrode solution. The modified Tyrode solution (pH 7.4)

contained the following (in mM): NaCl 135, KCl 4.0, MgCl2 1.0, HEPES 10, NaH2PO4 0.33,

glucose 10, butanedione monoxime 10, and the solution was gassed with 5% CO2-95% O2. The

digested heart was then removed from the cannula and left ventricle was cut into small pieces in the

modified Tyrode solution. Tissue pieces were gently agitated and pellet of cells was resuspended.

Extracellular Ca2+ was added incrementally back to 1.20 mM over a period of 30 min. Isolated

myocytes were used for experiments within 8 hours of isolation. Only rod-shaped myocytes with

clear edges were selected for mechanical and intracellular Ca2+ studies (3).

Cell shortening/relengthening

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Mechanical properties of ventricular myocytes were assessed using a SoftEdge MyoCam®

system (IonOptix Corporation, Milton, MA, USA) (3). In brief, cells were placed in a Warner

chamber mounted on the stage of an inverted microscope (Olympus, IX-70) and superfused (∼1

ml/min at 25oC) with a buffer containing (in mM): 131 NaCl, 4 KCl, 1 CaCl2, 1 MgCl2, 10 glucose,

10 HEPES, at pH 7.4. The cells were field stimulated with suprathreshold voltage at a frequency of

0.5 Hz (unless otherwise stated), 3 msec duration, using a pair of platinum wires placed on opposite

sides of the chamber connected to a FHC stimulator (Brunswick, NE, USA). The myocyte being

studied was displayed on the computer monitor using an IonOptix MyoCam camera. An IonOptix

SoftEdge software was used to capture changes in cell length during shortening and relengthening.

In the case of altering stimulus frequency from 0.1 Hz to 5.0 Hz, the steady state contraction of

myocyte was achieved (usually after the first 5-6 beats) before PS was recorded. Cell shortening and

relengthening were assessed using the following indices: peak shortening (PS), time-to-90% PS

(TPS90), time-to-90% relengthening (TR90), half width duration (HWD) – indicating late cell

shortening and early relengthening, as well as maximal velocity of shortening (+ dL/dt) and

relengthening (-dL/dt). To examine the acute effect of IGF-1 on cardiomyocyte contractile function,

cohorts of cardiomyocytes from young and aged FVB mice were treated with recombinant IGF-1

(500 nM) for 2 hrs at 37oC with 100% humidity and 5% CO2 as described previously (14).

Western blot analysis

Protein levels of SRECA2a, PLB, NCX, AGE and protein carbonyl were examined by

standard Western blot. Left ventricular tissues were homogenized and centrifuged at 70,000 x g

for 20 min at 4°C. The supernatants were used for immunoblotting of SERCA2a, PLB, NCX,

AGE and carbonyl. The extracted proteins were separated on 10 - 15% SDS-polyacrylamide gels

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and transferred to polyvinylidene difluoride membranes. After blocking the membrane was

incubated with mouse anti-AGE monoclonal (1:1000, Trans Genic Inc., Kumamoto, Japan),

rabbit anti-NCX polyclonal (1:1000, Swant, Bellinzona, Switzerland) and mouse anti-PLB

monoclonal antibody (1:2000, Abcam Inc, Cambridge, MA), rabbit anti-SERCA2a (1:1000,

Affinity BioReagents Inc., Golden, CO) and rabbit anti-DNP (dinitrophenyl, 1:150, Chemicon

International, Temecula, CA) overnight at 4°C followed by incubation with second antibodies.

The antigens were detected by the luminescence method. Quantification of band density was

determined using Quantity One software (Bio-Rad, version 4.4.0, build 36) and reported in

optical density per square millimeter (16).

SERCA activity measured by 45Ca2+ uptake

Cardiomyocytes were sonicated and solubilized in a Tris-sucrose homogenization buffer

consisting of 30 mM Tris-HCl, 8% sucrose, 1 mM PMSF and 2 mM dithioithreitol, pH 7.1. To

determine SERCA-dependent Ca2+ uptake, samples were treated with and without the SERCA

inhibitor thapsigargin (10 µM) for 15 min. The difference between the two readings was deemed

the thapsigargin-sensitive uptake through SERCA. Uptake was initiated by the addition of an

aliquot of supernatant to a solution consisting of (in mM) 100 KCl, 5 NaN3, 6 MgCl2, 0.15

EGTA, 0.12 CaCl2, 30 Tris-HCl pH 7.0, 10 oxalate, 2 ATP and 1 µCi 45

CaCl2 at 37°C. Aliquots

of samples were injected onto glass filters on a suction manifold and washed 3 times. Filters

were then removed from the manifold, placed in scintillation fluid and counted. SERCA activity

was expressed as cpm/mg protein (15).

Statistical analysis

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Data were presented as Mean ± SEM. Statistical significance (p < 0.05) for each variable

was determined by analysis of variance (ANOVA) or t-test, where appropriate.

RESULTS

General feature of young and old experimental animals

General features of young and aged FVB and IGF-1 transgene mice are shown in Table 1.

At young age, transgenic IGF-1 overexpression did not elicit any notable effect on body, liver and

kidney weights compared with age-matched FVB mice. However, IGF-1 significantly increased

heart weight and heart size (heart-to-body weight ratio) in young mice. The enlarged heart weight

and size seen in young IGF-1 mice prevailed through the older age. Aged mice had heavier body

and organ weights (although not organ size) compared with young counterparts with the exception

of a large kidney size. Aging significantly reduced plasma IGF-1 levels. IGF-1 overexpression

significantly elevated plasma IGF-1 levels in young mice and nullified aging-induced decline in

plasma IGF-1 levels. Fasting blood glucose levels were not affected by either IGF-1 transgene or

age.

Mechanical properties of cardiomyocytes from young or aged FVB and IGF-1 mice

Mechanical properties obtained at a pace frequency of 0.5 Hz revealed that resting cell

length and PS amplitude were similar in ventricular myocytes among young and aged FVB and

IGF-1 mice with the exception of a reduced resting cell length in aged IGF-1 mice. However,

myocytes from aged FVB mice displayed significantly reduced maximal velocity of

shortening/relengthening (± dL/dt) compared with myocytes from young FVB mice. The reduced ±

dL/dt was associated with normal shortening duration (TPS90), prolonged relengthening duration

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(TR90) and half-width duration (HWD) in aged FVB myocytes. Interestingly, these aging-induced

mechanical dysfunctions were not observed in aged IGF-1 transgenic mice, suggesting a beneficial

role of IGF-1 against aging-associated cardiac dysfunction. IGF-1 itself did not affect mechanical

contractile properties in young mice. To explore if IGF-1 transgene-elicited beneficial effect against

cardiac aging was due to IGF-1-induced cardiac hypertrophy or positive inotropicity as described

previously (20-22), cohorts of cardiomyocytes from young and aged FVB mice were treated in vitro

with recombinant IGF-1 (500 nM) for 2 hrs before mechanical property was recorded. Our data

revealed that IGF-1 treatment alleviated aging-induced decrease in ± dL/dt, prolongation in TR90

and HWD, in a manner similar to endogenous IGF-1 overexpression. In vitro IGF-1 treatment did

not significantly affect cardiomyocyte mechanical function in young mice with the exception of

significantly shortened HWD (Fig. 1).

Effect of increasing stimulation frequency on myocyte shortening

To evaluate the impact of aging on cardiac contractile function under various frequencies,

stimulus frequency was raised from 0.1 Hz to 5.0 Hz and the steady-state peak shortening was

recorded. Cells were initially stimulated to contract at 0.5 Hz for 5 min to ensure steady-state

before commencing the frequency response study. All recordings were normalized to PS at 0.1

Hz of the same myocyte. Fig. 2 shows a steeper negative staircase in PS in aged FVB myocytes

with increased stimulus frequency (at 0.5 Hz or higher) compared with young FVB myocytes,

suggesting reduced stress intolerance under advanced aging. Interestingly, IGF-1 transgene

ablated aging-induced stepper reduction in PS amplitude at high stimulating frequencies.

Protein expression of intracellular Ca2+ regulatory proteins, AGE and carbonyl formation

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As depicted in Fig 3, Western blot analysis demonstrated a significantly reduced expression

of SERCA2a in aged FVB group which was nullified by IGF-1. IGF-1 itself did not affect the

expression of SERCA2a. Neither aging nor IGF-1 affected expression of Na+-Ca2+ exchanger and

phospholamban (monomer). IGF-1 but not aging significantly reduced the expression of pentamer

phospholamban. Our data further revealed that significantly elevated formation of AGE and protein

carbonyl in aged FVB mouse hearts. IGF-1 attenuated aging-elicited elevation of protein carbonyl

but not AGE. Interestingly, IGF-1 itself significantly reduced the levels of AGE and protein

carbonyl in young mouse hearts (Fig. 4).

Effect of IGF-1 on SERCA activity in young and aged mice

Consistent with reduced SERCA2a protein expression in aged FVB mouse hearts shown

in Fig. 3, direct measurement of SERCA activity using 45Ca uptake technique revealed that aging

significantly dampened SERCA activity which may be rescued by IGF-1. Similar to its effect on

SERCA2a protein expression, IGF-1 transgene itself did not affect SERCA activity in young

FVB mouse hearts (Fig. 5).

DISCUSSION

Our study revealed that cardiac overexpression of IGF-1 rescued aging-induced cardiac

contractile abnormalities. The IGF-1-induced cardiac protection against aging-induced cardiac

defects may be underscored by alteration in SERCA protein expression and function as well as

protein carbonyl formation. Furthermore, cardiac-specific overexpression of IGF-1 helped to

alleviate aging-induced decline in plasma IGF-1 levels. Since IGF-1 itself did not significantly

affect cardiac contractile function in young mice, its protective role against aging-induced

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cardiac dysfunction may implicate certain clinical potential of this growth factor in delaying

cardiac aging process and minimize senescence-associated high cardiovascular mortality.

An ample of evidence has confirmed dysregulated cardiac function in senescence (4; 9;

13). Our results revealed reduced maximal velocity of contraction and relaxation, prolongation of

relaxation and half-width duration in aged FVB cardiomyocytes. We found that IGF-1 attenuated

aging-induced cardiac contractile dysfunction, SERCA expression/function and protein carbonyl

formation, indicating possible contribution of facilitated intracellular Ca2+ re-sequestration and

reduced protein damage from IGF-1 transgene. The fact that IGF-1 itself did not affect myocyte

function, SERCA expression and function in young mouse hearts indicates the excessive amount

of this growth factor is not innately harmful to cardiac function. It was demonstrated previously

that IGF-1 overexpressing transgenic mice exhibit protection against diabetes-induced cardiac

pathology, oxidative damage, activation of oncogene p53 and cardiac contractile dysfunction (7;

18). IGF-1 is known to trigger cardiac hypertrophy (20; 22), as evidenced by significantly

increased cardiac mass and heart-to-body weight ratio (Table 1). It is possible that the IGF-1

overexpression-associated changes in cardiomyocyte function and protein expression under

aging could be consequences of cardiac hypertrophy. Nonetheless, data from our short-term in

vitro treatment of recombinant IGF-1 indicated that IGF-1-induced cardiac hypertrophy is less

likely responsible for the IGF-1-elicited protective effect against cardiac aging. The observation

that short-term IGF-1 incubation did not significantly affect young cardiomyocyte contractile

function (with the exception of shortened HWD) is consistent with our previous report (14),

indicating a possibly sufficient IGF-1 levels in young cardiomyocytes. The fact that recombinant

IGF-1 short-term treatment shortened HWD without affecting TPS90 and TR90 in young

cardiomyocytes indicates that IGF-1 may predominantly facilitate late phase contraction and

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early phase relaxation (which comprises HWD). However, such response possibly elicited by

acute IGF-1 treatment may be adapted with sustained high IGF-1 levels in IGF-1 overexpression

condition.

SERCA and NCX are considered as the main machineries to remove Ca2+ from cytosolic

space for cardiac relaxation to occur. However the relative contribution for intracellular Ca2+

extrusion by SERCA and NCX are still debatable, and may vary among different species (1; 5;

12). Bers indicated that SERCA is responsible for 92% Ca2+ removal while NCX only

contributes to <10% in mouse hearts (1). Our data revealed down-regulation of SERCA but not

NCX or phospholamban, an endogenous inhibitor of SERCA, in aged FVB hearts. The reduced

SERCA2a protein expression is supported by reduced SERCA activity in aged FVB hearts,

which may account for, at least in part, diastolic dysfunction (prolonged relaxation and half-

width duration) in aged myocytes. One rather surprising finding from our study was that IGF-1

transgene reduced phospholamban pentamer expression without affecting that of phospholamban

monomer. Although no precise mechanism regarding IGF-1-elicited response on phospholamban

subunit can be offered at this time, two scenarios may be considered. First, the phosphorylation

status of phospholamban may affect the affinity of phospholamban antibody (6). Although our

earlier study did not reveal phosphorylation of phospholamban with short-term treatment of IGF-

1 (14), the potential that sustained IGF-1 overexpression alters phospholamban phosphorylation

and thus antibody affinity for unphosphorylated phospholamban cannot be ruled out. Secondly, it

was depicted that phospholamban monomer rather than pentamer is deemed the “active” SERCA

inhibitor (23). Therefore, the IGF-1-elicited downregulation on phospholamban pentamer may be

non-specific and trivial to the overall effect of IGF-1 on SERCA function, given that

phospholamban monomer is unaffected by IGF-1.

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IGF-1 is a peptide growth factor structurally and functionally similar to insulin. It is

synthesized by various cell types including cardiomyocytes and acts as an autocrine/paracrine

factor (20; 22). IGF-1 regulates myocardial growth and function under both physiological and

pathophysiological conditions. It improves myocardial function in post-infarction rat heart, in

patients with chronic heart failure and in healthy humans and experimental animals (2; 17; 22).

Advanced age is associated with insufficient production of GH levels and hepatic GH resistance.

This reduction in GH secretion is sufficient to cause a fall in the serum IGF-1 level, abnormal body

composition and metabolism (19). These changes are distinct from those associated with the

hyposomatotropism of the elderly, but are less severe than those seen in younger adults with organic

GH deficiency. Evidence has indicated that GH and/or IGF-I are involved in the regulation of

cardiovascular function. Patients with GH/IGF-1 deficiency displayed comparable cardiac

dysfunctions to aging, manifested primarily as reduced left ventricular mass, ejection fraction, and

diastolic filling. IGF-1 is believed to mediate, to a large extent, the action of GH in cardiac

growth and contraction. It improves cardiac contractility, tissue remodeling, glucose metabolism,

insulin sensitivity and lipid profile (22). Data from our present study suggest that cardiac-specific

overexpression of IGF-1, which may be secreted from cardiomyocytes into circulation (7; 20),

may compensate for the reduced circulating IGF-1 levels and subsequently impaired SERCA

expression/function under aging.

In conclusion, our study revealed that cardiac specific overexpression of IGF-1 rescues

aging-induced cardiomyocyte mechanical dysfunction possibly through improvement in SERCA

expression and function, in addition to its anti-protein damage capacity. It should be stated that

other known beneficial effect of IGF-1 including anti-oxidant property should not be ruled out at

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this time. These data have convincingly demonstrated the clinical potential of IGF-1 in the

prevention and treatment of aging-associated cardiac dysfunction.

ACKNOWLEDGMENTS

The authors would like to thank Bonnie H. Zhao and Nicholas S. Aberle II for their

assistance in data acquisition and analysis. This work was supported in part by National Institute

of Health grants to JR (NIA: 1 R03 AG21324-01 and NIAAA: 1R15 AA13575-01). This work

was presented in the 15th World Congress of Pharmacology in Beijing, P.R. China in July 2006.

No disclosure is declared from any of the authors.

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FIGURE LEGENDS

Fig. 1: Contractile properties of cardiomyocytes from young or old FVB and IGF-1 transgenic

mice. A cohort of cardiomyocytes from young and old FVB mice was treated in vitro with IGF-1

(500 nM) for 2 hrs prior to assessment of mechanical function. A: Resting cell length; B: Peak

shortening (PS, normalized to resting cell length); C: Half-width duration (HWD); D: Maximal

velocity of shortening/relengthening (± dL/dt); E: Time-to-90% PS (TPS90); and F: Time-to-90%

relengthening (TR90). Mean ± SEM, n = 62-64 cells/group, * p < 0.05 vs. FVB young group, # p

< 0.05 vs. FVB-old group.

Fig. 2: Peak shortening (PS) amplitude of cardiomyocytes from young or old FVB and IGF-1

mice at different stimulus frequencies (0.1 – 5.0 Hz). PS was shown as % change from PS at 0.1

Hz of the same cell. Mean ± SEM, n = 38 – 40 cells per group, * p < 0.05 vs. FVB young group,

# p < 0.05 vs. FVB-old group.

Fig. 3: Western blot analysis exhibiting expression of SERCA2a, Na+-Ca2+ exchanger (NCX)

and phospholamban (PLB) in ventricles from young and old FVB and IGF-1 mice. A: SERCA2a

expression; B: Na+-Ca2+ exchanger expression, C: phospholamban monomer expression; and D:

phospholamban pentamer expression. Inset: Represent gel blots depicting expression of above

mentioned proteins using specific antibodies. Mean ± SEM, n = 6 -8 samples per group, * p <

0.05 vs. FVB young group, # p < 0.05 vs. FVB-old group.

Fig. 4: Western blot analysis exhibiting expression of AGE (panel A) and protein carbonyl

(Panel B) in ventricles from young and old FVB and IGF-1 mice. Inset: Represent gel blots

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depicting expression of above mentioned proteins using specific antibodies. Mean ± SEM, n = 5

-8 samples per group, * p < 0.05 vs. FVB young group, # p < 0.05 vs. FVB-old group.

Fig. 5: SERCA2 activity measured by SERCA-dependent 45Ca2+ uptake in ventricles from young

and old FVB and IGF-1 mice. Mean ± SEM, n = 6, * p < 0.05 vs. FVB young group, # p < 0.05

vs. FVB-old group.

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Table I: General features of young- and old-FVB and IGF-1 mice.

FVB-young FVB-old IGF-1-young IGF-1-old

Body Weight (g) 19.1 ± 0.5 28.7 ± 0.8* 20.7 ± 0.9 30.2 ± 0.6*

Heart Weight (mg) 98 ± 4 156 ± 8* 126 ± 6** 185 ± 10*,**

HW/BW (mg/g) 5.12 ± 0.18 5.31 ± 0.22 6.12 ± 0.17** 6.19 ± 0.19**

Liver Weight (g) 0.89 ± 0.03 1.51 ± 0.07* 0.98 ± 0.05 1.40 ± 0.06*

LW/BW (mg/g) 47.6 ± 1.4 51.3 ± 1.2 47.6 ± 1.3 46.1 ± 1.4

Kidney Weight (g) 0.23 ± 0.01 0.42 ± 0.02* 0.25 ± 0.02 0.45 ± 0.02*

KW/BW (mg/g) 12.2 ± 0.4 14.6 ± 0.6* 12.1 ± 0.4 15.0 ± 0.5*

Blood glucose (mg/dl) 97.0 ± 5.6 99.1 ± 6.4 95.2 ± 6.4 100.8 ± 7.0

Plasma IGF-1 (ng/ml) 101.8 ± 6.0 48.6 ± 4.2* 154.4 ± 5.3** 87.3 ± 5.7**

HW = heart weight; LW = liver weight; KW = kidney weight; Mean ± SEM, * p < 0.05 vs.

corresponding young group, ** p < 0.05 vs. corresponding FVB group n = 17 – 23 mice/group.

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Li et al., Fig. 1

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Li et al., Fig. 3

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Li et al., Fig. 4

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1.0

1.5

2.0

FVB-Young

FVB-Old

IGF-O

ld

IGF-Y

oung

*

#

Pro

tein

Car

bo

nyl

(Arb

itra

ryD

en

sity

)

B.

AGE100 KD50 KD37 KD

Protein Carbonyl

98 KD

64 KD

50 KD

36 KD

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FVB-Young

1SE

RC

A a

ctiv

ity-

-[45

]Ca2+

up

take

(CP

M/ m

g p

rote

in/ 3

0 m

in)

0

2000

4000

6000

8000

10000

12000

FVB-Old

IGF-Y

oung

IGF-O

ld

*

Li et al., Fig. 5

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