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Am J Physiol Heart Circ Physiol 291: H787-H796, 2006. First published March 24, 2006; doi:10.1152/ajpheart.00974.2005
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Hyperinsulinemia: effect on cardiac mass/function, angiotensin II receptor expression, and insulin signaling pathways

Anne-Maj Samuelsson, Entela Bollano, Reza Mobini, Britt-Mari Larsson, Elmir Omerovic, Michael Fu, Finn Waagstein, and Agneta Holmäng

Cardiovascular Institute and Wallenberg Laboratory, Sahlgrenska University Hospital,Göteborg University, Göteborg, Sweden

Submitted 11 September 2005 ; accepted in final form 7 March 2006


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
To investigate the association between hyperinsulinemia and cardiac hypertrophy, we treated rats with insulin for 7 wk and assessed effects on myocardial growth, vascularization, and fibrosis in relation to the expression of angiotensin II receptors (AT-R). We also characterized insulin signaling pathways believed to promote myocyte growth and interact with proliferative responses mediated by G protein-coupled receptors, and we assessed myocardial insulin receptor substrate-1 (IRS-1) and p110{alpha} catalytic and p85 regulatory subunits of phospatidylinositol 3 kinase (PI3K), Akt, MEK, ERK1/2, and S6 kinase-1 (S6K1). Left ventricular (LV) geometry and performance were evaluated echocardiographically. Insulin decreased AT1a-R mRNA expression but increased protein levels and increased AT2-R mRNA and protein levels and phosphorylation of IRS-1 (Ser374/Tyr989), MEK1/2 (Ser218/Ser222), ERK1/2 (Thr202/Tyr204), S6K1 (Thr421/Ser424/Thr389), Akt (Thr308/Thr308), and PI3K p110{alpha} but not of p85 (Tyr508). Insulin increased LV mass and relative wall thickness and reduced stroke volume and cardiac output. Histochemical examination demonstrated myocyte hypertrophy and increases in interstitial fibrosis. Metoprolol plus insulin prevented the increase in relative wall thickness, decreased fibrosis, increased LV mass, and improved function seen with insulin alone. Thus our data demonstrate that chronic hyperinsulinemia decreases AT1a-to-AT2 ratio and increases MEK-ERK1/2 and S6K1 pathway activity related to hypertrophy. These changes might be crucial for increased cardiovascular growth and fibrosis and signs of impaired LV function.

renin-angiotensin system; hypertrophy; mitogen-activated protein kinase; phosphatidylinositol 3 kinase

CARDIAC HYPERTROPHY and insulin resistance are closely associated pathophysiological findings (44, 50). In hypertensive patients, left ventricular (LV) hypertrophy (LVH) is associated with hyperinsulinemia and a decline in insulin-mediated glucose uptake (44, 50). Hypertension can alter LV geometry, resulting in concentric LVH [increased mass and relative wall thickness (RWTh)], eccentric LVH (increased mass, normal RWTh), or concentric remodeling (normal mass, increased RWTh). Patients with LVH, primarily concentric LVH, have the highest rates of all, which cause mortality and cardiovascular morbidity (15, 32). The relationship between LVH and blood pressure, however, explains only 25–30% of the variation in LV mass (45), suggesting the involvement of another hemodynamic trophic mechanism.

One potentially important factor is the growth effect of insulin (8). In concert with insulin, insulin-like growth factor 1 (IGF-1) (8) may stimulate vascular growth and increase cardiac mass. Signal transmission by the insulin and IGF-1 receptor (IGF-1R) to downstream effectors is largely mediated by insulin receptor substrate (IRS) adapter protein 1 (46). IRS-1 is primarily involved in somatic cell growth, protein synthesis, and insulin action in muscle and adipose tissue (46). These signaling pathways have different, but also overlapping, functions (46). After tyrosine phosphorylation, IRS proteins act as docking proteins for several proteins containing Src homology 2 domains, including phosphatidylinositol 3 kinase (PI3K), which is composed of a p110 catalytic subunit and p85, p55, or p50 regulatory subunits (46). A key effector of the PI3K pathway is a protein kinase termed protein kinase B (PKB), also known as Akt. Akt is activated through phosphorylation of the residues Thr308 and Ser473. Thr308 is in the core of the protein kinase catalytic domain in a region known as the activation loop, regulated by the protein kinase 3-phosphoinositide-dependent kinase-1 (PDK-1), whereas Ser473 is located in a carboxy terminal noncatalytic region known as the hydrophobic motif and phosphorylated by an unindentified kinase called PDK-2 (1, 2). Akt (Ser473) phosphorylation has been associated with insulin resistance (39). In response to insulin, Akt promotes GLUT 4-mediated glucose uptake and glucogen synthesis via serine phosphorylation (14, 30). Akt is also critical for the induction of cardiac hypertrophy through subsequent phosphorylation of several downstream pathways such as mitogen-activated protein kinases (MAPKs) and ribosomal S6 kinases (4, 36).

Ribosomal S6 protein is phosphorylated by p70 ribosomal S6 kinase (S6K1), a key factor for protein synthesis in various cell types (36). The S6K1 consist of two phosphorylation sites: the inhibitory domain (Thr421/Ser424) and the catalytic domain Thr389. The phosphorylation of S6K1 (Thr421/Ser424) is regulated by the PI3K pathway, whereas S6K1 (Thr389) is regulated by both PI3K and PDK-1 pathways (3). Both of these sites are activated in insulin-induced smooth muscle growth (31).

Rapamycin, a specific inhibitor of the mammalian target of rapamycin (mTOR), an upstream activator of S6K1, inhibits cardiac myocyte hypertrophy mediated by ANG II, phenylephrine, and isoprenaline (28, 47). Thus S6K1 is a key factor in the signal transduction pathway that regulates protein synthesis in the hypertrophic growth of cardiac myocytes, but it can also inhibit IRS proteins (62). Extracellular signal-regulated kinase (ERK) 1/2, a well-studied MAPK activated by its upstream kinase MAPK-extracellular signal-regulated kinase (MEK), is important for cell growth and differentiation (21, 62). ERK 1/2 may be stimulated by neurohumoral factors (52) and mechanical stress (63) as well as by insulin and is considered essential for cardiac myocyte hypertrophy (22, 43).

ANG II, the biologically active component of the renin-angiotensin system (RAS), is important in the development of cardiomyocyte hypertrophy and cardiac fibrosis. ANG II acts through two subtypes of receptors: AT1-R and AT2-R. AT1-R increases norepinephrine release from sympathetic nerve terminals, the rate and force of cardiac contraction, and myocardial cell growth (59). AT1-R antagonists reduce cardiac hypertrophy in animal models (41) and in hypertensive patients (7), demonstrating the role of AT1-R in LVH. Although the biological actions of ANG II were once believed to be mediated largely by the AT1-R, recent investigations established a role for the AT2-R in cardiovascular function, development, and apoptosis and perhaps in mediating growth-inhibiting effects in the heart (35).

Insulin stimulates the RAS signaling pathway and induces overexpression of AT1-R, thereby enhancing the biological efficacy of ANG II (38). Both insulin and ANG II have growth-promoting effects that appear to be mediated by interactions among insulin, AT-1, and AT-2 receptors that stimulate MAPK-ERK1/2, S6K1, and PI3K-Akt pathways in target cells such as myocytes and fibroblasts (38, 60).

Long-term studies of insulin exposure do not seem to have controlled for increased secretion of catecholamine (53) and adrenal hormones (11). We have previously used an experimental approach in which rats are exposed to prolonged, physiological hyperinsulinemia with counterregulatory factors by adrenalectomy, beta-adrenergic blockade, and glucocorticoid supplementation. In this model, insulin sensitivity increases (24, 26). With the use of this method, the effects of insulin per se on myocardial growth may be examined after long-term exposure without the development of insulin resistance and associated factors, which counteract insulin effects.

Thus the goal of this study was to assess the effects of chronic hyperinsulinemia on myocardial growth and fibrosis in relation to expression of ANG II receptors (AT-R1a, AT-R2) in rats. To gain further insight into the effects of hyperinsulinemia on the insulin signaling pathways that might interact with hypertrophic responses mediated by G protein-coupled receptors, we assessed the phosphorylation levels of IRS-1, p110{alpha} catalytic and p85 regulatory subunits of PI3K, and Akt1, MEK, ERK1/2, and S6K1. In addition, we wanted to examine the functional effects on LV geometry and performance by using echocardiographic measurements.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. Female Sprague-Dawley rats (9 wk old, 230 ± 8 g) (B&K Universal, Sollentuna, Sweden) were fed on standard rat pellets and tap water ad libitum. Standard principles of laboratory animal care were followed. All procedures were approved by the Animal Ethics Committee of Göteborg University.

Study groups. There were four experimental groups and two control groups (n = 10 each). Experimental rats were treated with insulin (INS), insulin and adrenalectomy (INS+ADX), insulin and metoprolol (INS+MET), or INS+ADX+MET. Control rats were treated with sterile saline (NaCl) or sterile saline and adrenalectomy (NaCl+ADX). Adrenalectomy was performed at the same time as the osmotic minipumps were implanted (10). All ADX animals were substituted with cortisol phosphate (400 µg·kg–1·day–1; Solu-Cortef, Upjohn, Puurs, Belgium) diluted in saline and given as a daily subcutaneous injection at 0800 (5).

Study design. The rats were intubated and ventilated with 0.5 l/min air, 1.5 l/min O2, and 2.5 l/min Isofluran (Abbot Laboratories, Abbott Park, IL), and osmotic minipumps 2ML4 (2.5 µl/h, Alza, Palo Alto, CA) containing either metoprolol (5 mg·kg–1·h–1; Sigma-Aldrich, Stockholm, Sweden) and/or insulin (2 U/day, at a rate of 1 µl/h; Hoe-21 PH; Hoechst, Frankfurt am Main, Germany) were implanted subcutaneously as described and changed after 3 wk. After 7 wk of treatment, the rats were anesthetized, their hearts were quickly excised, and the atria, ventricles, and septum were dissected and fixed in 4% paraformaldehyde or frozen in liquid nitrogen (–80°C). The right tibia of each rat was isolated, and its length was measured.

Echocardiography and hemodynamics. After 7 wk, LV geometry and function were assessed by two-dimensional, M-mode, and Doppler echocardiography (42). All measurements represent the mean of three consecutive cardiac cycles, determined with the leading-edge method (48). Systolic arterial pressure was measured with a tail-cuff monitor (RTBP Monitor, Harvard Apparatus, South Natick, MA) after weeks 1, 3, 5, and 7. In each rat, mean systolic arterial pressure (MSAP) and heart rate (HR) were calculated from the average of three consecutive recordings.

Analytical methods. After 7 wk of treatment, nonfasting tail blood samples were collected. The plasma glucose concentration was determined with an autoanalyzer by using the glucose oxidase method (YSI Scientific, Yellow Springs, OH). Commercial assay kits were used to determine the plasma levels of insulin (Linco Research, St. Charles, MO), insulin-like growth factor 1 (Mediagnost, Reutlingen, Germany), and triiodothryonine (ICN Biochemicals, Irvine, CA).

Histochemical methods. Tissue sections (5 µm) were taken from the middle portion of the lateral wall of the left ventricle and mounted on glass slides. Each section was stained with hematoxylin-eosin, Weigert's hematoxylin-van Gieson for collagen density (fibrosis) (Histocenter, Göteborg, Sweden), and {alpha}-sarcomeric actin staining for cardiomyocyte density, as described (24). All histological measurements were carried out on light microscopic images (BP 485/20, FT 510, LP 520; Carl Zeiss, Hallbergmoos, Germany) in a blinded manner. Ten random areas from each slide were selected with KS400 image-analysis software (Carl Zeiss).

Real-time PCR analysis. Total RNA from the atrium was extracted with RNeasy Mini kits (Qiagen, Hilden, Germany). PCR analyses were performed with the ABI Prism 7700 Sequence Detection System (PE Applied Biosystems, Stockholm, Sweden) by using FAM-labeled probes specific for the AT1a-R and AT2-R (PE Applied Biosystems). The reactions included designed primers; a VIC-labeled probe for beta-actin served as an internal standard. cDNA was amplified as follows: 1 cycle at 50°C for 2 min and 95°C for 10 min, followed by 40 cycles at 95°C for 15 s and 60°C for 1 min. mRNA was quantitated with the standard curve method (USerBulletin 2, PE Applied Biosystems) and adjusted for beta-actin expression.

Western blot analysis. Total protein extracts from frozen ventricles were prepared as described (40). The primary polyclonal antibodies were anti-AT1a-R (1:2,000) (Abcam, Cambridge, UK), anti-AT2-R (1:1,000), IGF-1R{alpha} (1:500), IRS-1 (1:200), anti-MEK (1:1,000), anti-ERK (1:1,000), PI3K p110{alpha} (1:2,000), and anti-beta-actin (1:2,000) (Santa Cruz Biotechnology, Santa Cruz, CA). Phosphospecific antibodies to PI3K p85{alpha} (Tyr508) (1:1,000), Akt1/2 (Ser473) (1:1,000), Akt1/2/3 (Thr308) (1:500), S6K1 (Thr421/Ser424) (1:500), S6K1 (Thr389) (1:500), MEK1/2 (Ser218/Ser222) (1:1,000), ERK1/2 (Thr202/Tyr204) (1:1,000), IRS-1 (Tyr989) (1:200), and IRS-1(Ser374) (1:200) were from Santa Cruz Biotechnology. All antibodies were diluted in Tris-buffered saline, pH 7.6, containing 0.1% Tween 20 (TBS-T). The secondary antibody was a horseradish peroxidase-conjugated anti-IgG (Amersham Pharmacia Biotech, Amersham, Buckinghamshire, UK) diluted 1:20,000 in TBS-T. After blotting was completed, nitrocellulose membranes were incubated at 50°C for 30 min in stripping buffer containing 2% SDS, 6.25 mM Tris·HCl, pH 6.8, and 0.704% (vol/vol) beta-mercaptoethanol, washed extensively with 0.01% Tween 20 and Tris-buffered saline, pH 7.0, and blocked with 5% dry milk for 1 h. Blots were reprobed with primary antibody overnight at 4°C. Immunoreactive proteins were visualized with the indicated primary antibodies and enhanced ECL reagents followed by autoradiography and densitometry.

Statistical analysis. Statistical comparisons were performed with Statview 5.0 (Abacus Concepts, Berkeley, CA). Three-factor ANOVA considering INS, ADX, and MET was used for intergroup comparisons. Additive treatment effects were analyzed by one-way ANOVA and Tukey's post hoc test. Relationships between variables were assessed with Pearson's correlation coefficient. All tests were two-tailed. Differences were considered significant at the 5% level.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Insulin increases body weight, tibia length, heart weight, and LV mass. All INS rats had a significantly higher total body weight (BW), tibia length (TL), and heart weight (HW) than controls (Table 1). Because insulin promotes the growth of bone and adipose tissues, TL and BW cannot be used to normalize HW measurements (61). LV mass was increased in all INS rats. MET+INS increased HW and LV mass, as did MET+INS+ADX, compared with insulin treatment only. Right ventricular (RV) mass was significantly higher in INS and INS+ADX rats than that in controls. MET treatment decreased the insulin effect on RV mass, relative to insulin treatment alone. BW correlated with LV mass (R = 0.45, P < 0.001, n = 60; 10/group) but not with RWTh (R = –0.14, P = 0.51, n = 48; 8/group).


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  		Table 1. Body weight, tibia length, heart weight, and LV and RV mass after 7 wk of treatment

 
Insulin-induced MSAP and HR activation. At 1 wk, MSAP was higher in INS rats (18 mmHg) than NaCl controls or rats treated with ADX and/or MET (Table 2). ADX and MET had additive effects on MSAP. HR was higher in the group treated with insulin alone than controls at weeks 3 and 5 and was reduced by treatment with ADX and/or MET.


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  		Table 2. MSAP and HR after 1, 3, 5, and 7 wk of treatment

 
Increased RWTh and impaired LV function. Echocardiographic examinations were performed after 7 wk of treatment (Table 3). Rats treated with INS or INS+ADX had lower ratios of LV diastolic diameter (LVDd) and LV systolic diameter (LVDs) to BW and higher RWTh than those in controls. The increases in LV mass (Table 1) and RWTh indicate concentric hypertrophy. Addition of MET increased LV diameter (LVDd/BW, LVDs/BW) with no change in RWTh; this, together with the increased LV mass, indicates eccentric hypertrophy. RWTh correlated with plasma insulin levels (R = 0.79, P < 0.001, n = 48; 8/group) but not with MSAP (R = –0.23, n = 48; 8/group).


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  		Table 3. Cardiac dimensions and function after 7 wk of treatment

 
The decreased LV diameter (LVDd and LVDd/BW) in rats treated with insulin also resulted in decreased stroke volume (SV) (SV/BW) without any change in fractional shortening (FS). Consequently, cardiac output (CO) (CO/BW) was reduced. MET significantly increased SV/BW compared with insulin alone; however, CO/BW was similar in these groups. ADX had no significant effect on the echocardiographic findings.

Plasma insulin and glucose levels. Nonfasting plasma glucose levels did not differ between groups (mean: 7.4 ± 0.3 mmol/l, n = 60; 10/group). The plasma insulin levels were significantly elevated in INS animals (133 ± 9 µU/l, n = 40; 10/group) compared with NaCl and NaCl+ADX animals (73.3 ± 8 µU/l, n = 20; 10/group, P < 0.01). Plasma IGF-I and T3 were not affected by INS, MET, or INS+MET (not shown).

LV fibrosis increases. Cardiomyocyte and collagen (fibrosis) after 7 wk of treatment are shown in Table 4. Interstitial fibrosis was higher in the INS, INS+ADX, and INS+ADX+MET groups than that in controls. MET significantly reduced insulin-induced fibrosis compared with insulin alone. Myocyte density was decreased in all INS rats except for the INS+ADX group, and myocyte cross-sectional area increased by a similar magnitude. Thus the cardiac hypertrophy indicated by the increased HW in INS rats can be explained by an increase in myocyte size.


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  		Table 4. Cardiomyocyte and total collagen (fibrosis) from lateral wall of left ventricle after 7 wk of treatment

 
Altered AT1a-R and AT2-R mRNA and protein levels. Atrial levels of AT1a-R and AT2-R mRNA and ventricle levels of AT1a-R and AT2-R are shown in Fig. 1. The control groups had higher expression of AT1a-R than those of AT2-R mRNA (5:1). The reverse was true of the INS group (1:2) and other INS rats. All INS rats had significantly lower expression of AT1a mRNA and higher expression of AT2 mRNA than controls. However, the AT2 levels did not differ in INS+ADX+MET group compared with controls. AT1a and AT2 protein levels were increased only in the INS group. There was no difference in beta-actin expression between the groups, thus used as an endogen control.


Figure 1
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  		Fig. 1. Angiotensin II receptors AT1a-R mRNA (A) and AT2-R mRNA (B) of the left atrium and AT1a-R protein (C) and AT2-R protein (D) of the left ventricle. Representative blots are shown under each bar graph. All insulin (INS)-treated rats had lower AT1a-to-AT2 mRNA ratio compared with controls. AT1a and AT2 protein levels were increased in INS group only. ADX, adrenolectomy, MET, metoprolol. ***P < 0.001, **P < 0.01, *P < 0.05 vs. NaCl and NaCl+ADX. {ddagger}{ddagger}P < 0.01, {ddagger}P < 0.05 vs. INS (one-way ANOVA). Values are means ± SE; n = 8 rats.

 
Insulin increases serine IRS-1 and decreases tyrosine IRS-1 phosphorylation. LV levels of IRS-1 (Tyr989) and IRS 1 (Ser374) are shown in Fig. 2. Tyrosine phosphorylation IRS-1 (Tyr989) was decreased in all INS groups, except INS+ADX groups. Serine phosphorylation IRS-1 (Ser374) was increased in INS and INS+MET compared with NaCl. There was no significant difference in total IRS-1 between the different groups.


Figure 2
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  		Fig. 2. Phosphorylated INS receptor substrate-1 (IRS-1) (Tyr989) (A) and IRS-1 (Ser374) (B) protein levels in left ventricle. Representative blots are shown under each bar graph. Serine IRS-1 phosphorylation was increased in INS and INS+MET, tyrosine IRS-1 phosphorylation was decreased in all INS-treated groups, except INS+ADX+MET. ***P < 0.001, **P < 0.01, *P < 0.05 vs. NaCl; {ddagger}{ddagger}P < 0.01, {ddagger}P < 0.05 vs. INS (one-way ANOVA). Values are means ± SE; n = 8 rats.

 
Increased activity in MEK-ERK1/2, PI3k P110{alpha}, and S6K1 pathways but not PI3K P85. LV levels of phosphorylated MEK1/2 (Ser218/Ser222), ERK1/2 (Thr202/Tyr204), PI3K-p85 (Tyr508), p110, Akt (Thr308), Akt (Ser473), S6K1 (Thr421/Ser424), and S6K1 (Thr389) are shown in Fig. 3. In the INS group, MEK1/2 (Ser218/Ser222) and ERK1/2 (Thr202/Tyr204) levels were significantly higher than the levels in controls (Fig. 3, A and B). Additive treatment with MET and/or ADX reduced MEK1/2 (Ser218/Ser222) and ERK1/2 (Thr202/Tyr204) protein to control levels. The PI3K p110{alpha} subunit was increased in the INS group without any significant difference in PI3K p85 (Tyr508) subunit (Fig. 3, C and D). The Akt (Thr308) protein levels were increased in all INS groups, without any significant differences in Akt (Ser473) levels (Fig. 3, E and F). The S6K1 (Thr421/Ser424) and S6K1 (Thr389) phosphorylation levels were significantly higher in all INS groups compared with controls (Fig. 3, G and H). There were no significant differences in the levels of total MEK and ERK between the different groups (results not shown).


Figure 3
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  		Fig. 3. Phosphorylated MEK1/2 (Ser218/Ser222) (A), ERK1/2 (Thr202/Tyr204) (B), phosphatidylinositol 3 kinase (PI3K) p85 (Tyr508) (C), PI3K p110{alpha} (D), Akt (Ser473) (E), Akt (Thr308) (F), S6 kinase-1 (S6K1) (Thr421/Ser424) (G), and S6K1 (Thr389) (F) protein levels in left ventricle. Representative blots are shown under each bar graph. Phosphorylated MEK1/2 (Ser218/Ser222) and ERK1/2 (Thr202/Tyr204) were increased in INS group. PI3k p110{alpha} was increased in the INS group and Akt (Thr308) levels were increased in all INS-treated animals. S6K1 (Thr421/Ser424) and S6K1 (Thr389) levels were increased in all the INS-treated animals. **P < 0.01, *P < 0.05 vs. NaCl; {ddagger}{ddagger}P < 0.01, {ddagger}P < 0.05 vs. INS (one-way ANOVA). Values are means ± SE; n = 8 rats.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
This study shows that chronic hyperinsulinemia increased the protein AT1- and AT2-R levels; these effects were prevented by the addition of ADX and/or MET to insulin treatment. Hyperinsulinemia also increased the serine phosphorylation of IRS-1 (Ser374) (as did INS+MET), MEK1/2 (Ser218/Ser222), ERK1/2 (Thr202/Tyr204), S6K1 (Thr421/Ser424), S6K1 (Thr389), and PI3K p110{alpha} and decreased tyrosine phosphorylation IRS-1 (Tyr989) (except INS+ADX), whereas the p85 regulatory subunit of PI3K (Tyr508) was not altered. Hyperinsulinemia also caused myocyte hypertrophy and increased interstitial fibrosis. Concomitantly, LV mass and RWTh increased, and LV geometry was altered, leading to signs of impaired LV function; the impairment was prevented by treatment with a selective beta1-antagonist or by blocking glucocorticoid release with ADX. These findings point to a central role for insulin in the regulation, triggering, and interactions of signaling pathways and genes important for myocardial growth and function in vivo.

Hyperinsulinemic rat model. Insulin resistance and hyperinsulinemia are two components of a cluster of cardiovascular and metabolic risk factors known as the metabolic syndrome. Owing to their frequent coexistence, it is difficult to identify their importance, both individually and in combination, inrelation to LV mass and LV function. We used an experimental model in which rats were rendered hyperinsulinemic while counterregulatory factors were controlled and plasma glucose levels were kept within the normal range. In our previous (24, 25) and recent studies (unpublished observations), insulin sensitivity was unaltered in INS rats. Thus we could examine the effects of insulin per se on cardiac function and gene expression and on the development of cardiac hypertrophy and remodeling in the absence of hyperglycemia and associated factors that counteract or alter the effects of insulin.

Angiotensin receptors and hyperinsulinemia. Insulin treatment increased AT2-R mRNA expression (except when ADX and MET were added) and decreased AT1-R mRNA expression in the atrium. Conversely, AT1-R and AT2-R protein levels in the ventricle were increased only in rats treated with insulin alone. The INS group had also the highest density of fibrosis, suggesting that interstitial fibroblasts are the major cell type responsible for AT2-R expression. This possibility is consistent with the likelihood that AT2-R expression in the human heart failure is determined by the extent of interstitial fibrosis, because fibroblasts in interstitial regions are the major cell type responsible for this enhanced expression (33).

In the embryonic heart, the AT2-R is highly expressed. In the adult heart, however, AT1-R expression is predominant, and the AT2-R is expressed at low levels (29). This adult pattern is consistent with the AT1-R-to-AT2-R mRNA and protein ratios in the control groups. Insulin treatment induced a changed pattern of gene expression, as observed in pathologically altered tissues and conditions, and is strongly connected to cardiac myocyte hypertrophy (29). Interestingly, the AT1-R is regulated predominately by posttranscriptional mechanisms (19, 37). Insulin induces AT1-R overexpression, which might be mediated by an effect that protects the receptor from posttranscriptional degradation. Importantly, the additional AT1-Rs are functional (38). However, the role of the insulin-induced increase in AT2-R levels, which also changed the ratio between the two ANG II receptors, must be further elucidated.

Hyperinsulinemia and insulin signaling. Insulin treatment alone increased the serine phosphorylation of IRS-1 (Ser374) (as did INS+MET) and decreased tyrosine phosphorylation (Tyr989) (except INS+ADX) without altering the phosphorylation of the p85 regulatory subunit of PI3K (Tyr508), but it increased the activities of the p110{alpha} catalytic unit of PI3K, MEK1/2 (Ser218/Ser222), and ERK1/2 (Thr202/Tyr204). Conversely, phosphorylation of S6K1 increased in all insulin-exposed rats. Thus insulin stimulation per se is important in both the MEK-ERK1/2 and S6K1 pathways, which regulate protein synthesis in the hypertrophic growth of cardiac myocytes. Interestingly, the ERK inhibitor PD-98059 attenuated cardiac-specific gene expression in a dose-dependent manner but had no effect on protein synthesis or cell size mediated by the S6K1 pathway (43). This is in accordance with our hypothesis that insulin induces cardiac hypertrophy through different and probably at least partly independent but additive pathways of regulation.

The threonine phosphorylation Akt (Thr308) was increased in all insulin-exposed rats, whereas serine phosphorylation Akt (Ser473) remained unchanged. Consistent results have been shown in smooth muscle cells from INS animals, suggesting two different upstream pathways, either the PDK1-Akt (Thr308) or PI3p85-Akt (Ser473) (55). Previous studies have indicated decreased serine phosphorylation Akt (Ser473) with insulin resistance (9, 39). Our model, however, showed no effect on insulin sensitivity (24, 25, and unpublished data) and consequently no effect on Akt (Ser473) phosphorylation.

Two different pathways also regulate the relationship between Akt and MAPK. Inhibition of p38MAPK (SB-203580) showed a dose-dependent inhibition of Akt serine phosphorylation (Ser473) but not (Thr308), whereas ERK1/2 inhibitor PD-98059 shows no effect on serine phosphorylation Akt (Ser473) (55). Taken together, site-specific phosphorylation pathways regulate Akt; our model showed insulin induced Akt (Thr308) phosphorylation in an ERK1/2-dependent manner.

Cellular processes such as differentiation and growth are influenced by the integration of both synergistic and antagonistic stimuli. Glucocorticoids and IGF-1 reciprocally modulate growth-regulated processes (e.g., translation initiation) by inactivating S6K1 in skeletal muscle in vitro (49). In a recent study, ADX per se did not affect protein synthesis rates in rats; however, glucocorticoid deficiency enhanced the insulin sensitivity of muscle protein synthesis after 3 h of insulin clamping through increased phosphorylation of proteins such as S6K1 that regulate translation initiation (34). Although we also found that S6K1 phosphorylation was not affected by ADX, it was not increased by the addition of insulin. This discrepancy may reflect differences in the cell types studied or the longer insulin exposure time (7 wk) in our study, which can lead to an adaptation.

Insulin alone increased serine phosphorylation of IRS-1 (Ser374) (as did INS+MET) but not of the regulatory subunit p85 of PI3K (Tyr508) or Akt (Ser473) in either group, whereas p110{alpha} protein level was increased in the group treated with insulin alone. The tyrosine phosphorylation, known to activate IRS proteins, was found to be modest but significantly decreased in our study (except INS+ADX), whereas the phosphorylation of IRS-1 on serine residues, which has a dual role to either enhance or terminate the insulin effects (23), was increased in the INS and INS+MET groups. The role of serine phosphorylation has not been fully understood but seems to enhance the growth effects of insulin because the increase in myocyte size was most pronounced in these two groups in this study.

Physiological control of PI3K signaling probably involves negative feedback that attenuates the strength or duration of PI3K activation, as occurs in other signaling pathways (36). A primary mechanism shown in a recent study is the inhibition of IRS protein function through the phosphorylation of serine residues that disconnects the insulin receptor from PI3K-Akt activation (6). The discrepancy between PI3K activity and Akt activity may reflect p85-dependent negative regulation of downstream PI3K signaling (58). However, the p110{alpha} isoform of PI3K, which couples to tyrosine kinase receptors, is critical in regulating heart growth (51), and a finely tuned balance probably exists between the positive signal from the p110-IRS complex and the negative signal from the p85 subunit.

Recently, another pathway that regulates PI3K was identified, suggesting that a major form of negative feedback inhibition of PI3K p85 results from activated growth signaling via mTOR and S6K1 (57). Importantly, this effect, which was induced by acute insulin exposure of L6 muscle cells, correlated temporally with full activation of S6K1, providing the first indication of negative feedback control of PI3K p85 activity. However, the kinase might not only decrease or dampen the activity of the p85 but also stimulate the p110{alpha} isoform as seen in our study. Futhermore, in response to insulin, S6K1 is not solely dependent on the PI3K pathway. Recent studies showed that intracellular Ca2+ levels activate S6K1 independently of PI3K/Akt (13, 31). This might explain the discrepancy between S6K1 and PI3K activation.

Hyperinsulinemia and AT-Rs. In our study, AT1-R and AT2-R protein levels were significantly upregulated only in the group treated with insulin alone, the only group exhibiting significantly increased phosphorylation of proteins involved in the MEK-ERK1/2 pathway. In vitro studies have demonstrated both transactivation of receptor tyrosine kinase by mitogenic G protein-coupled receptors (16) and functional transinhibition due to reduced autophosphorylation of the insulin receptor and increased serine phosphorylation of IRS-1 protein (18).

Interestingly, there seems to be direct cross-talk between insulin and the ANG II signaling pathways at the level of both IRS-Grb2 association and ERKs (12, 20). Such cross-talk promotes cardiac myocyte growth (17). ERK1/2 is activated by ANG II through the AT1-R in an IRS-dependent fashion or by insulin through the insulin receptor (60). In rats, injection of ANG II acutely inhibited both basal and insulin-stimulated PI3K activity; this effect was blocked by AT1-R antagonists (60). In another study, PI3K and MEK-ERK1/2 pathways were compared in cardiac tissue of control rats and obese, insulin-resistant Zucker rats, in which insulin signaling was impaired at the level of PI3K (12). Insulin injection stimulated activation of ERK1/2 equally in the two groups, and pretreatment with the AT1-R blocker losartan did not change this activation. Conversely, insulin-induced stimulation of Akt decreased in the obese rats but not in the controls, and losartan attenuated this reduction. Despite the inhibition of PI3K activity, ANG II stimulates cellular hypertrophy through both the MEK-ERK1/2 and S6K1 pathways (56). In accordance with these studies, cross-talk between ANG II and insulin signaling might occur at several independent levels, which also seems to be the case in our study.

LV performance and hyperinsulinemia. Our echocardiographic findings suggest that insulin induces concentric LVH, whereas INS+MET induces eccentric LVH; both effects were independent of ADX. INS groups that developed concentric LVH also had signs of impaired pump function and reductions in LVDd and LVDs. As a result, both SV and CO were reduced. MET prevented the impairment or even improved pump function, despite the eccentric hypertrophy. Insulin also seemed to be a powerful determinant of RWTh and concentric LVH, consistent with studies showing that circulating insulin levels are more closely related to RWTh than to LV mass (27, 54). In our study, RWTh did not correlate with BW or MSAP, but LV mass correlated with BW. These finding suggest that different factors mediate the effects of LV mass and RWTh and that insulin alone has a strong remodeling effect on the LV. RV mass was also increased in the INS rats. However, an increase in MSAP does not directly affect RV mass, and there is no other reason to believe that there is an increased pressure load on the RV. Thus the hypertrophy we observed probably reflects a nonhemodynamic growth effect of insulin. This possibility merits further exploration.

Perspectives. Our findings in this study point to a central role for insulin in the regulation, triggering, and interactions of signaling pathways and genes important for myocardial growth and function in vivo. The challenge for future studies will be to further elucidate the mechanisms involved, which might be of clinical relevance for new therapeutic strategies.


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This work was supported by the Swedish Medical Research Council Project No. 12206 and the Swedish Heart Lung Foundation.


   FOOTNOTES
 

Address for reprint requests and other correspondence: A.-M. Samuelsson, The Wallenberg Laboratory, Sahlgrenska Univ. Hospital, Göteborg Univ., S-413 45 Göteborg, Sweden (E-mail: anne-maj.samuelsson{at}wlab.gu.se)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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