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The Journals of Gerontology Series A: Biological Sciences and Medical Sciences 63:788-797 (2008)
© 2008 The Gerontological Society of America

Insulin Signaling Cascade in the Hearts of Long-Lived Growth Hormone Receptor Knockout Mice: Effects of Calorie Restriction

Jorge F. Giani, Michael S. Bonkowski, Marina C. Muñoz, Michal M. Masternak, Daniel Turyn, Andrzej Bartke and Fernando P. Dominici

1 Instituto de Química y Fisicoquímica Biológicas (IQUIFIB), Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Argentina.
2 Geriatrics Research, Departments of Internal Medicine and Physiology, and 3 Department of Pharmacology, School of Medicine, Southern Illinois University, Springfield.

Address correspondence to Fernando Pablo Dominici, PhD, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Instituto de Química y Fisicoquímica Biológicas (IQUIFIB), Junin 956, sexto piso, Buenos Aires, Buenos Aires 1113, Argentina. E-mail: dominici{at}qb.ffyb.uba.ar


   Abstract
Top
 Abstract
Methods
 Results
 Discussion
 References
 
Calorie restriction (CR) improves insulin sensitivity and increases life span in normal but not in long-lived growth hormone-resistant knockout (GHRKO) mice. In this study, we examined interactive effects of GH resistance and long-term CR on cardiac insulin action. GHRKO mice exhibited marked increases in the insulin-induced phosphorylation of the insulin receptor (IR), insulin receptor substrate-1 (IRS-1), Akt, and ERK1/2 along with elevated insulin-stimulated IRS-1-associated regulatory subunit of phosphatidylinositol 3-kinase in the heart. These changes were associated with elevated protein levels of IR, IRS-1, and Akt and with a down-regulation of cardiac glucose transporter 4 (GLUT4). In normal mice, CR induced an important increase in the phosphorylation of cardiac Akt without elevation of Akt protein, reaching activation levels similar to those seen in GHRKO mice. This change may be cardioprotective and thus contribute to increased longevity in response to CR. Interestingly, the insulin signaling cascade in the heart of GHRKO mice was unaffected by CR.

Key Words: GHRKO mice • Calorie restriction • Heart • Insulin signaling

INSULIN signaling influences numerous functions within the heart, such as metabolic substrate preference, cell size, and the response of the heart to ischemia and hypertrophy (1–3). Growth hormone (GH) exerts anti-insulinemic effects (4–6) and exerts complex dose-related effects on cardiac function, with both GH deficiency and GH excess believed to be important risk factors for cardiovascular disease (7–9). Deletion of GH receptor gene in Ghr/GHbp –/– mice (hereafter referred to as GHRKO mice) leads to GH resistance, hypoinsulinemia, enhanced whole-animal insulin sensitivity as shown in an intraperitoneal insulin tolerance test, and remarkable increases in longevity (10–13). These animals have very low levels of circulating insulin-like growth factor-1 (IGF-1), reduced body size, an elevated percentage of adiposity with major differences in fat distribution (14,15), and decreased systolic blood pressure and plasma renin (16), and they exhibit various indices of delayed aging (17–19).

Reduced food intake, also known as calorie restriction (CR), produces a robust increase in the health and longevity of mice, rats, and other species (20,21). Many effects of CR resemble phenotypic characteristics of GHRKO mice (18). These characteristics include hypoinsulinemia and improved insulin sensitivity. We have recently demonstrated that subjecting male GHRKO mice to 30% CR starting at 2 months of age does not produce further increases in their life span or insulin sensitivity (except for a modest increase in maximal, but not median or average longevity) (19). Our previous studies indicate that CR exerts differential and organ-specific effects on the expression of genes related to insulin signaling in the liver, skeletal muscle, and heart of GHRKO mice as compared to their normal siblings (22–24).

Objectives of the present study were (i) to compare the impact of GH receptor deletion and CR on the effects of acute in vivo insulin stimulation on early steps of cardiac insulin signaling, and (ii) to compare the effects of CR on cardiac insulin signaling in GHRKO mice to CR effects in normal animals.


   METHODS
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 Abstract
 Methods
Results
 Discussion
 References
 
Materials
The reagents and apparatus for sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and immunoblotting were obtained from Bio-Rad (Hercules, CA). The monoclonal antiphosphotyrosine antibody (anti-p-Tyr, PY99), the rabbit polyclonal anti-insulin receptor (IR) β subunit antibody (anti-IR, C-19), the rabbit polyclonal antiglucose transporter (GLUT)1 antibody (H-43), the goat antirabbit immunoglobulin G (IgG) conjugated with horse radish peroxidase (HRP), and goat antimouse IgG-HRP secondary antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The polyclonal anti-insulin receptor substrate-1 antibody (anti-IRS-1), the rabbit polyclonal anti-GLUT4, and the antibody to the p85{alpha} subunit of phosphatidylinositol 3-kinase (PI3K) (anti-p85) were purchased from Upstate Biotechnology (Lake Placid, NY). Antiphospho-Akt (Ser473) mouse monoclonal antibody, the polyclonal Akt antibody that detects endogenous levels of total Akt1, Akt2, and Akt3 proteins (anti-Akt); the polyclonal antiphospho-ERK1/2 antibody that detects endogenous levels of ERK1 and ERK2 (p44 and p42 mitogen-activated protein [MAP] kinase, respectively) when phosphorylated at Thr 202 and Tyr 204; and the polyclonal anti-ERK1/2 antibody were purchased from Cell Signaling (Beverly, MA). The anti-β-actin antibody and remaining reagents were purchased from Sigma-Aldrich (St. Louis, MO).

Animal Care and Feeding
The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health (NIH Publication No. 85-23, revised 1996), and the experimental protocol was approved by the Southern Illinois University Laboratory of Animal Care and Use Committee. GHRKO and normal mice were produced in our breeding colony derived from GHRKO animals provided by Dr. J. J. Kopchick (Ohio University, Athens OH). Phenotypically normal siblings of GHRKO mice served as controls for this study. Animals were housed under temperature- and light-controlled conditions (20–23°C and 12-hour light/dark cycle). At 8 weeks of age, mice matched for average body weight within phenotype were divided into two treatment groups: CR or fed ad libitum (AL). AL animals had constant access to food (Lab Diet Formula 5001, not autoclaved, 23.4% protein, 4.5% fat, 5.8% crude fiber; purchased from Lab Diet, PMI Feeds, Inc., St. Louis, MO). Animals subjected to CR were placed on 30% CR gradually by receiving 90% of the amount of food consumed by AL controls in the initial week, 80% the following week, and 70% throughout the remainder of the study (19). This experimental design resulted in eight groups of animals: normal (N)-AL, N-CR, GHRKO-AL and GHRKO-CR, each comprising saline- or insulin-stimulated mice. All groups of animals were given tap water AL.

Acute Insulin Stimulation and Tissue Collection
At the age of 14 months, after 12 months of dietary treatment, mice were starved overnight, and 10 minutes before the experiment they were anesthetized with isoflurane (Baxter Pharmaceutical Products Inc., Deerfield, IL). After anesthesia was induced, the inferior vena cava was exposed and 10 IU of porcine insulin per kilogram of body weight in normal saline (0.9% NaCl) in a final volume of 0.1 mL was injected via this vein as described previously (25). To obtain data under basal conditions, GHRKO and control mice received an injection of diluent. Approximately 3 minutes after injection, the heart was removed and kept at –80°C until analysis.

Tissue Homogenization
Tissue samples were homogenized in solubilization buffer containing 1% Triton together with phosphatase and protease inhibitors as described previously (25,26). Heart extracts were centrifuged at 100,000 x g for 1 hour at 4°C to eliminate insoluble material, and protein concentration in the supernatants was measured using the Bradford method as described previously (25,26).

Immunoprecipitation and Western Blotting Analysis
Equal amounts of solubilized heart protein (2 mg) were incubated at 4°C overnight with anti-IR or anti-IRS-1 antibodies at a final concentration of 4 µg/mL. Immune complexes were collected by incubation with protein A-Sepharose 6 MB as described previously (25,26). SDS–PAGE and Western transfer of proteins to polyvinylidene difluoride membranes were performed as previously described (25,26). Membranes were blocked by incubation for 2 hours with Tris-buffered saline containing 0.1% Tween 20 and 3% bovine serum albumin and subsequently incubated overnight with anti-p-Tyr (1:1000) to detect tyrosine phosphorylation. Membranes were reblotted with anti-IR (1:200) or anti-IRS-1 (1:1000) to determine protein abundance of these two proteins. To determine the amount of the p85 subunit of PI3K associated with IRS-1, membranes corresponding to anti-IRS-1 immunoprecipitates were also reprobed with anti-p85 antibody (1:2000). To determine the phosphorylation levels of Akt and ERK1/2 and the total protein abundance of GLUT4 and GLUT1, equal amounts of solubilized proteins (40 µg) were denatured by boiling in reducing sample buffer, resolved by SDS–PAGE, and subjected to immunoblotting with antiphospho-Akt, antiphospho-ERK1/2, anti-GLUT4, or anti-GLUT1 (1:1000 dilution for all antibodies). Cardiac Akt and ERK1/2 abundance were detected by reprobing the corresponding membranes with the anti-Akt or anti-ERK1/2 antibodies. After extensive washing, membranes were incubated with the appropriate secondary HRP-coupled antibodies and processed for enhanced chemiluminescence using the ECL plus Western Blotting detection system (Amersham Biosciences, Piscataway, NJ). Bands were quantified using Gel-Pro Analyzer 4.0 (Media Cybernetics, Inc., Bethesda, MD). Protein loading in gels was evaluated by Coomassie blue staining and by reblotting membranes with anti-β-actin antibody (1:10,000).

Statistical Analysis
Results are presented as mean ± standard error of the mean (SEM). Analysis of variance (ANOVA) was used to assess significance of differences. When ANOVA revealed significant differences, Tukey–Kramer correction for post hoc t tests was used to correct for multiple comparisons. A value of p <.05 was considered significant.


   RESULTS
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 Abstract
 Methods
 Results
Discussion
 References
 
Tyrosine Phosphorylation and Protein Levels of IR and IRS-1
Basal IR tyrosine phosphorylation values were very low in all four groups of animals (Figure 1A). Acute in vivo insulin administration induced a 2.2-fold increase in the tyrosine phosphorylation of the IR in the heart of normal animals (Figure 1A), whereas the same stimulus induced a 5.8-fold increase in the phosphorylation of the IR in GHRKO mice (p <.05 vs normal mice; n = 4; Figure 1A). The IR response to in vivo insulin administration was not altered by CR, regardless of phenotype (Figure 1A). Insulin receptor protein content was increased 3.3-fold in GHRKO mice (p <.05; n = 8; Figure 1B). CR did not induce significant changes in the content of IR in the heart from either normal or GHRKO mice (Figure 1B). When compared to diluents, IR phosphorylation was increased to the same extent as the increased receptor protein expression in GHRKO mice, suggesting that the increase in tyrosine-phosphorylated IR may have been a consequence of increased IR protein (Figure 1C).


Figure 01
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  		Figure 1. Insulin receptor (IR) tyrosine phosphorylation and protein content in heart of normal (N) or growth hormone–resistant knockout (KO) mice fed ad libitum (AL) or exposed to calorie restriction (CR). Hearts were removed after injection with insulin (+) or its diluent (–) into the portal vein. Equal amounts of solubilized heart protein were immunoprecipitated with an antibody to the β-subunit of the IR (anti-IR), run on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) gels, and immunoblotted (IB) with anti-p-Tyr. A, Basal and insulin-stimulated tyrosine phosphorylation of IR (n = 4 per group). *p <.05 vs the corresponding basal value; **p <.01 vs N-AL basal; #p <.05 vs N-AL insulin-stimulated. B, IR protein levels as detected by reprobing the membranes with anti-IR (n = 8 per group). *p <.05 vs N-AL. C, Ratio of the IR phosphorylation to IR protein content (n = 4 per group). Bars represent the means ± standard error of the mean

 
CR did not alter total IRS-1 protein or insulin-induced IRS-1 phosphorylation in normal mice (Figure 2, A and B). In GHRKO mice fed AL or CR, IRS-1 total protein levels were significantly elevated (p <.05; n = 8). In addition, IRS-1 tyrosine phosphorylation was observed to increase 4.2-fold in the heart of GHRKO mice compared with a 2.1-fold increase in the heart of normal animals (p <.05; n = 8; Figure 2A). CR did not modulate insulin responses at this level (Figure 2A). Furthermore, elevated insulin-stimulated IRS-1 tyrosine phosphorylation was accompanied by a 2.2-fold increase in the IRS-1 protein content (p <.05; n = 4; Figure 2B). The phosphorylation/IRS-1 protein ratio was not significantly different between the GHRKO and control mouse hearts, suggesting that the increase in IRS-1 protein was responsible for the increase in insulin-stimulated tyrosine phosphorylation of IRS-1 (Figure 2C).


Figure 02
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  		Figure 2. Insulin receptor substrate-1 (IRS-1) tyrosine phosphorylation and protein content in heart of normal (N) or growth hormone–resistant knockout (KO) mice fed ad libitum (AL) or exposed to calorie restriction (CR). Equal amounts of solubilized heart protein obtained as described in
Figure 1 were immunoprecipitated with an anti-IRS-1 antibody, run on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) gels, and immunoblotted (IB) with anti-p-Tyr. A, Basal and insulin-stimulated tyrosine phosphorylation of IRS-1 (n = 4 per group). *p <.05 vs the corresponding basal value; **p <.01 vs N-AL basal; #p <.05 vs N-AL insulin-stimulated. B, IRS-1 protein levels as detected by reprobing the membranes with anti-IRS-1 (n = 8 per group). *p <.05 vs N-AL. C, Ratio of IRS-1 phosphorylation to IRS-1 protein content (n = 4 per group). Bars represent the means ± standard error of the mean

 
Insulin-Stimulated IRS-1-Associated p85 Regulatory Subunit of PI3K
Tyrosine-phosphorylated IRS-1 associates with the regulatory subunit of PI3K (p85), resulting in PI3K activity induction. In all groups of animals, insulin stimulation induced an increase in the association of p85 with IRS-1 (p <.05; n = 4; Figure 3A). Compared with normal animals, GHRKO mice exhibited a robust and significant increase in the association of p85 with IRS-1 in response to insulin stimulation (4.2-fold vs 2.1-fold increase; p <.05; n = 4; Figure 3A). IRS-1–associated p85 was not modified after CR regardless of phenotype. Quantitation of multiple experiments revealed that the protein content of p85 in the heart was similar in all cases (Figure 3B), suggesting that the increased insulin-stimulated association between IRS-1 and p85 in the heart of GHRKO mice could be the result of the elevated amount of IR and IRS-1 total protein and the likely elevated protein induced activation seen in these experiments.


Figure 03
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  		Figure 3. Association of insulin receptor substrate-1 (IRS-1) with the p85 subunit of phosphatidylinositol 3-kinase (PI3K) and total p85 protein content in heart of normal (N) or growth hormone–resistant knockout (KO) mice fed ad libitum (AL) or exposed to calorie restriction (CR). Equal amounts of solubilized heart protein obtained as described in
Figure 1 were either immunoprecipitated with an antibody to IRS-1, run on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) gels, and immunoblotted (IB) with an anti-p85 antibody (A) or directly immunoblotted with the anti-p85 antibody (B). A, Basal and insulin-stimulated values for each of the four groups. Bars represent the means ± standard error of the mean (SEM) (n = 4 per group). *p <.05 vs the corresponding basal value; **p <.01 vs N-AL basal; #p <.05 vs N-AL insulin-stimulated. B, Protein abundance of p85. Data are the means ± SEM of four independent experiments (n = 8 per group)

 
Akt Serine Phosphorylation and Protein Levels
As shown in Figure 4A, Akt phosphorylation in the AL GHRKO mice was clearly increased when compared to insulin-stimulated normal values. Akt phosphorylation increased by 1.8-fold in control animals following insulin administration compared with a 4.1-fold increase in the heart of GHRKO mice (p <.05; n = 4; Figure 4A). Contrary to what was observed for IR and IRS-1, CR markedly up-regulated the insulin-stimulated Akt phosphorylation in normal mice (p <.05), whereas the same dietary regimen did not modify the already elevated insulin-stimulated Akt phosphorylation in GHRKO mice (Figure 4A). When compared to that in normal animals, the level of total Akt protein in GHRKO mice was increased by 100% (p <.05; n = 8), suggesting that the increase of insulin-induced Akt phosphorylation in GHRKO mice was a consequence of an increase in the expression of Akt (Figure 4B). However, Akt protein content in normal animals was not affected by CR, indicating that this dietary treatment increased the proportion of Akt protein that was phosphorylated (Figure 4C).


Figure 04
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  		Figure 4. Akt phosphorylation and protein content in heart of normal (N) or growth hormone–resistant knockout (KO) mice fed ad libitum (AL) or exposed to calorie restriction (CR). Equal amounts of solubilized heart protein obtained as described in
Figure 1 were run on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) gels and immunoblotted (IB) with the indicated antibodies. A, Basal and insulin-stimulated values for each of the four groups (n = 4 per group). *p <.05 vs N-AL basal; **p <.01 vs the corresponding basal value; #p <.05 vs N-AL insulin-stimulated. B, Acute insulin stimulation did not modify the abundance of Akt. Data are the means ± standard error of the mean (SEM) of four independent experiments (n = 8 per group). *p <.05 vs N-AL. C, Ratio of Akt phosphorylation to Akt protein content. Bars represent the means ± SEM (n = 4 per group). *p <.05 vs N-AL

 
GLUT4 and GLUT1 Protein Levels
The total amount of the glucose transporters GLUT4 and GLUT1 was determined by subjecting heart extracts to immunoblotting with anti-GLUT4 or anti-GLUT1. Content of GLUT4 protein was decreased by almost 65% in GHRKO mice fed AL (p <.05; n = 8, Figure 5A). CR did not modify the amount of GLUT4 in either group of animals (Figure 5A). Unlike what was observed for GLUT4, GHRKO mice displayed cardiac GLUT1 levels that were similar to those found in normal animals. Long-term CR did not affect the abundance of GLUT1 in either normal or knockout mice (n = 8, Figure 5B). The total amount of protein loaded in the gels was controlled by reprobing the same membranes with anti-β actin (Figure 5).


Figure 05
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  		Figure 5. Glucose transporter (GLUT)4 and GLUT1 content in heart of normal (N) or growth hormone–resistant knockout (KO) mice fed ad libitum (AL) or exposed to calorie restriction (CR). Equal amounts of solubilized heart protein obtained as described in
Figure 1 were run on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) gels and immunoblotted (IB) with anti-GLUT4 or anti-GLUT1 antibody. Typical blots show total amount of cardiac GLUT4 (A) and GLUT1 (B). The amount of protein loaded in the gels was controlled by reblotting the same membranes with an anti-β actin antibody (bottom). Acute insulin stimulation did not modify the abundance of the glucose transporters. Bars show the means ± standard error of the mean of four independent experiments (n = 8 per group). *p <.05 vs N-AL

 
ERK1/2 Phosphorylation and Protein Levels
Given that the MAP kinase cascade plays an essential role in cardiac myocyte growth, maintenance, and survival, we further examined the interaction between the effects of GH resistance and CR in the heart by measuring the insulin-stimulated and basal phosphorylation of ERK1/2 in vivo. Interestingly, when compared the values to values found in normal mice, GHRKO mice displayed a large increase in the insulin-stimulated phosphorylation of ERK1/2. In the heart, insulin administration induced a 3.9-fold increase in ERK1/2 phosphorylation in GHRKO mice compared with a 1.9-fold increase in normal mice (p <.05; n = 4; Figure 6A). Long-term CR did not affect the insulin-stimulated phosphorylation levels of these proteins in either normal or knockout mice (Figure 6A). The protein content of ERK1/2 was similar in all groups of animals (n = 8; Figure 6B), suggesting that the increased insulin-stimulated ERK1/2 phosphorylation in the heart of GHRKO mice reflected an increment in the activity of these proteins rather than an increase in their expression.


Figure 06
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  		Figure 6. ERK1/2 phosphorylation and protein content in heart of normal (N) or growth hormone–resistant knockout (KO) mice fed ad libitum (AL) or exposed to calorie restriction (CR). Equal amounts of solubilized heart protein obtained as described in
Figure 1 were run on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) gels and immunoblotted (IB) with an antiphospho ERK1/2 antibody (anti-p-ERK1/2). Protein content of ERK1/2 was determined by reblotting the same membranes with the anti-ERK1/2. A, Basal and insulin-stimulated values. Bars show the mean ± standard error of the mean (SEM) (n = 4 per group). *p <.05 vs the corresponding basal value; **p <.01 vs N-AL basal; #p <.05 vs N-AL insulin-stimulated. B, Acute insulin stimulation did not modify the abundance of ERK1/2. Bars represent the mean ± SEM of four independent determinations (n = 8 per group). C, Ratio of the ERK1/2 phosphorylation to ERK1/2 protein content. Data are expressed as means ± SEM (n = 4 per group). *p <.05 vs N-AL

 

   DISCUSSION
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 Abstract
 Methods
 Results
 Discussion
References
 
The key novel findings of the present study included evidence for major alterations in insulin signaling in the heart of long-lived, hypoinsulinemic GHRKO mice and differential effects of CR on insulin-induced Akt phosphorylation in the hearts of normal and GHRKO mice. Compared to the levels measured in normal mice, the levels and tyrosine phosphorylation of IR, IRS-1, and IRS-1-associated p85 subunit of PI3K, and the content and phosphorylation of Akt and ERK1/2 phosphorylation, were significantly increased in GHRKO mice.

Another novel finding of the present study was that long-term 30% CR did not affect any of the examined parameters of insulin signaling in the heart, except for phosphorylation of Akt at serine 473, which was increased by CR in normal but, interestingly, not in GHRKO mice.

Previous work from this and other laboratories provided evidence that the state of hypersensitivity to insulin exhibited by GHRKO mice coexists with increased IR protein levels and insulin-stimulated IR phosphorylation in the liver and with delayed and/or diminished responses in IR and IRS-1 phosphorylation in skeletal muscle, pointing to the liver as a major contributor to increased whole-animal insulin sensitivity in these animals (11,27). The heart is also a target of insulin (1,28). In vivo insulin administration leads to activation of canonical insulin signaling components in this organ (29). The phenotype of cardiomyocyte-selective IR knockout mice indicated that insulin signaling plays an important developmental role in regulating postnatal cardiac size, myosin isoform expression, and the switching of cardiac substrate utilization from glucose to fatty acids (2). Moreover, recent studies have suggested that adequate myocardial insulin sensitivity is essential to the maintenance of normal cardiovascular function (30).

The present results show that GHRKO mice, which have a phenotype of severe GH resistance, exhibit a marked increase in the in vivo insulin-induced stimulation at several steps within the insulin signaling system in the heart, suggesting that GH signaling is dominant in counteracting insulin's actions in the heart. Because we have previously shown that the expression of IR and IRS-1 messenger RNA is unaltered in the heart of GHRKO mice (23), we postulate that the elevation in the protein levels of IR and IRS-1 proteins resulted from a post-transcriptional mechanism. Hypoinsulinemia (reduction of insulin levels to 20%–40% of normal values) is a salient feature of GHRKO mice that has been consistently found in our previous studies (10,11). This characteristic has also been reported by other groups (12). In most studies, both IR and IRS-1 levels have been shown to be inversely related to insulin levels in vivo (31,32). Thus, we postulate that the increase in the IR and IRS-1 abundance in the heart most likely reflects up-regulation of these two molecules in response to hypoinsulinemia. These changes could then lead to compensatory increases in downstream molecules, explaining our current results.

Increased levels of AKT protein and insulin-stimulated phosphorylation of Akt in the heart of GHRKO mice could represent cardioprotection. Akt is a powerful survival signal in many systems, and is activated by several cardioprotective ligand-receptor systems, including insulin and IGF-1 (33). In addition, previous studies have established that the PI3K/Akt signaling pathway plays an important role in cardiac function by mediating postnatal growth in this organ (34,35).

GH and its local effector IGF-1 are important for normal growth of the heart and for maintaining cardiac mass and function (7,36). Compromised ventricular function and increased cardiovascular risk have been observed in individuals with GH deficiency (8,9). The cardiac features of GHRKO mice have been recently reported, and this phenotype is consistent with the effects of GH resistance described in the clinical syndrome of Laron dwarfism (16). Cardiac weight and volume were reduced in GHRKO mice to a larger extent than could be explained by the lower body weight of these animals (16). However, despite a reduced global systolic function, the heart of GHRKO mice produces a resting cardiac output sufficient to supply the body at rest (16). In a condition of GH resistance and severely reduced circulating IGF-1 previously reported in GHRKO mice, the actions of insulin as a growth factor for the heart could become more relevant. The MAP kinase pathway is involved in the growth-promoting effects of insulin (37). Thus, the large increase in insulin-stimulated ERK phosphorylation detected in the GHRKO heart could represent an essential compensatory up-regulation that allows maintenance of normal cardiac growth and function in the absence of the actions of GH and/or reduced peripheral IGF-1.

Unlike what was observed for the activation of ERK, the expression of the insulin-sensitive glucose transporter GLUT4 was markedly reduced in the hearts of GHRKO mice. The energy metabolism of the heart is dominated by oxidation of fatty acids, which accounts for more than 80% of adenosine triphosphate production under physiological conditions. Whereas glucose is not a dominant fuel in the heart, its consumption is substantial when compared to other cell types. It assumes even greater importance under conditions of stress such as ischemic damage (1). Heart muscle expresses GLUT1 and GLUT4 in a 1:3 ratio (1). The relative functions of these transporters have been clarified by the generation of mice with cardiac-restricted GLUT4 deletion. Cardiac glucose uptake in these animals is markedly reduced, despite a 3-fold increase in GLUT1 expression (38), indicating that, although GLUT1 may be the major mediator of basal cardiac glucose uptake in quiescent cardiomyocytes, GLUT4 is a more important regulator of glucose uptake in the contracting heart and therefore critical for cardiac function. Thus, decreased expression of GLUT4 in the heart of GHRKO mice in the present study may be indicative of a compensatory down-regulation that may favor normal function of the heart in these animals in the face of an exaggerated response to insulin. Our observation that GLUT1 is unaltered in the heart of GHRKO mice gives greater emphasis to this hypothesis.

Restricting daily caloric intake in laboratory rodents increases life span and delays or prevents age-related pathophysiological changes (20). In humans, CR markedly improves cardiovascular disease risk factor profiles and reduces symptoms of aging in diastolic heart function (39–41). We have previously reported that, in contrast to its effects in normal mice, CR does not improve insulin sensitivity or increase longevity in GHRKO mice (19). In the present study, CR increased the insulin-stimulated phosphorylation of Akt at Ser473 in normal mice, without elevating total Akt protein, implying an exaggerated activation of this enzyme in response to insulin. Although this increase was similar to that measured in GHRKO as compared to normal mice under conditions of AL feeding, downstream ERK1/2 activation was not elevated in normal mice on long-term CR. In addition, GHRKO mice showed reduced GLUT4 protein content, whereas normal mice on long-term CR had unaltered GLUT4 protein content compared to controls. It has been previously shown that Akt is involved in activation and shuttling of GLUT4 to the cell surface in response to insulin activation (37). Thus, one could postulate that normal animals on long-term CR show increased Akt phosphorylation in response to insulin stimulation, further increasing insulin-stimulated glucose uptake.

CR did not alter the activation of any of the tested insulin-signaling peptides in the already insulin-sensitive GHRKO mice. In contrast to the effects of CR in normal mice, AL- and CR-fed GHRKO mice showed a reduction in GLUT4 and an increase in activated ERK1/2. This differential regulation of cardiac GLUT4 abundance and ERK1/2 activation could be triggered by further increases in insulin action in the GHRKO mice, leading to compensatory growth through MAP kinase signaling, rather than increased glucose uptake. This shift towards MAP kinase signaling would be consistent with the reductions of GH action and peripheral IGF-1 previously reported in GHRKO mice. An alternative hypothesis is that the increase in Akt activation without increased total Akt protein seen in normal mice on long-term CR may represent a cardioprotective effect. Previous studies have reported elevations in both the protein level and the phosphorylation of Akt in skeletal muscle in response to both short- and long-term CR (42,43). CR has a potential cardioprotective effect (40,44,45). In support of the hypothesis that increased Akt activation is cardioprotective, the enhanced response to insulin in terms of Akt phosphorylation was the only feature that was shared between normal mice on CR and GHRKO mice.

Conclusion
The positive modulation of the insulin-signaling cascade found in the hearts of GHRKO mice may contribute to the remarkable increase in life span of these animals. Differential effects of CR on cardiac Akt activation in normal and GHRKO mice suggest another potential mechanism for the selective effect of CR on longevity in normal but not in GHRKO mice.


   Acknowledgments
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 Results
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This work was supported by National Institute on Aging grants AG19899 and U19 AG023122, the Ellison Medical Foundation, and the Southern Illinois University Geriatrics Medicine and Research Initiative. F. P. Dominici and D. Turyn are Career Investigators from Consejo Nacional de Investigaciones Científicas y Tecnológicas of Argentina (CONICET) and received grant support from the University of Buenos Aires (UBA), CONICET, and Agencia Nacional de Promoción Científica y Tecnológica of Argentina. J. F. Giani is a research fellow from UBA, and M.C. Muñoz is a research fellow from CONICET.

We thank J. Panici, J. Rocha, K. Al-Regaiey, and Feiya Wang for their laboratory assistance. We also thank S. Sandstrom for editorial help.


   Footnotes
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Decision Editor: Huber R. Warner, PhD

Received February 18, 2008

Accepted April 28, 2008


   References
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