Inhibition of PI-3 kinase/Akt/mTOR, but not calcineurin signaling, reverses insulin-like growth factor I-induced protection against glucose toxicity in cardiomyocyte contractile function

in Journal of Endocrinology
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Shi-Yan Li Division of Pharmaceutical Sciences and Center for Cardiovascular Research and Alternative Medicine, University of Wyoming, Laramie, Wyoming 82071, USA

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Cindy X Fang Division of Pharmaceutical Sciences and Center for Cardiovascular Research and Alternative Medicine, University of Wyoming, Laramie, Wyoming 82071, USA

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Nicholas S Aberle II Division of Pharmaceutical Sciences and Center for Cardiovascular Research and Alternative Medicine, University of Wyoming, Laramie, Wyoming 82071, USA

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Bonnie H Ren Division of Pharmaceutical Sciences and Center for Cardiovascular Research and Alternative Medicine, University of Wyoming, Laramie, Wyoming 82071, USA

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Asli F Ceylan-Isik Division of Pharmaceutical Sciences and Center for Cardiovascular Research and Alternative Medicine, University of Wyoming, Laramie, Wyoming 82071, USA

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Jun Ren Division of Pharmaceutical Sciences and Center for Cardiovascular Research and Alternative Medicine, University of Wyoming, Laramie, Wyoming 82071, USA

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(Requests for offprints should be addressed to J Ren; Email: jren@uwyo.edu)
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Insulin-like growth factor-I (IGF-1) ameliorates cardiac dysfunction in diabetes although the mechanism of action remains poorly understood. This study examined the role of PI-3 kinase/Akt/mammalian target of rapamycin (mTOR) and calcineurin pathways in cardiac effects of IGF-1 against glucose toxicity. Adult rat ventricular myocytes were cultured for 8 h with either normal (NG, 5.5 mM) or high (HG, 25.5 mM) glucose, in the presence or absence of IGF-1 (10–500 nM), the PI-3 kinase/Akt inhibitor LY294002 (10 μM), the mTOR inhibitor rapamycin (20 μM) or the calcineurin inhibitors cyclosporin A (5 μM) or FK506 (10 mg/l). Mechanical properties were evaluated using an IonOptix MyoCam system. HG depressed peak shortening (PS), reduced maximal velocity of shortening/relengthening (± dl/dt) and prolongs time-to-90% relengthening (TR90), which were abolished by IGF-1 (100 and 500 nM). Interestingly, the IGF-1-elicited protective effect against HG was nullified by either LY294002 or rapamycin, but not by cyclosporine A or FK506. None of the inhibitors affected cell mechanics. Western blot analysis indicated that HG and IGF-1 stimulated phosphorylation of Akt and mTOR. HG also activated p70s6k and suppressed GSK-3β phosphorylation. However, the HG-induced alterations in phosphorylation of Akt, mTOR, p70s6k and GSK-3β were significantly reversed by IGF-1. Protein expression of Akt, mTOR, p70s6k, GSK-3β, SERCA2a and phospholamban was unaffected by HG, IGF-1 or rapamycin. Rapamycin significantly enhanced Akt phosphorylation whereas it inhibited mTOR phosphorylation. Collectively, our data suggest that IGF-1 may provide cardiac protection against glucose in part through a PI-3 kinase/Akt/mTOR/ p70s6k-dependent and calcineurin-independent pathway.

Abstract

Insulin-like growth factor-I (IGF-1) ameliorates cardiac dysfunction in diabetes although the mechanism of action remains poorly understood. This study examined the role of PI-3 kinase/Akt/mammalian target of rapamycin (mTOR) and calcineurin pathways in cardiac effects of IGF-1 against glucose toxicity. Adult rat ventricular myocytes were cultured for 8 h with either normal (NG, 5.5 mM) or high (HG, 25.5 mM) glucose, in the presence or absence of IGF-1 (10–500 nM), the PI-3 kinase/Akt inhibitor LY294002 (10 μM), the mTOR inhibitor rapamycin (20 μM) or the calcineurin inhibitors cyclosporin A (5 μM) or FK506 (10 mg/l). Mechanical properties were evaluated using an IonOptix MyoCam system. HG depressed peak shortening (PS), reduced maximal velocity of shortening/relengthening (± dl/dt) and prolongs time-to-90% relengthening (TR90), which were abolished by IGF-1 (100 and 500 nM). Interestingly, the IGF-1-elicited protective effect against HG was nullified by either LY294002 or rapamycin, but not by cyclosporine A or FK506. None of the inhibitors affected cell mechanics. Western blot analysis indicated that HG and IGF-1 stimulated phosphorylation of Akt and mTOR. HG also activated p70s6k and suppressed GSK-3β phosphorylation. However, the HG-induced alterations in phosphorylation of Akt, mTOR, p70s6k and GSK-3β were significantly reversed by IGF-1. Protein expression of Akt, mTOR, p70s6k, GSK-3β, SERCA2a and phospholamban was unaffected by HG, IGF-1 or rapamycin. Rapamycin significantly enhanced Akt phosphorylation whereas it inhibited mTOR phosphorylation. Collectively, our data suggest that IGF-1 may provide cardiac protection against glucose in part through a PI-3 kinase/Akt/mTOR/ p70s6k-dependent and calcineurin-independent pathway.

Introduction

Glucose toxicity contributes to development of diabetic cardiomyopathy characterized by systolic and diastolic dysfunctions independent of coronary macro- and micro-vascular diseases (Galderisi et al. 1991, Ren et al. 1997). Diastolic dysfunction is the most prominent cardiac abnormality and is characterized by prolonged relaxation and decreased compliance (Galderisi et al. 1991, Ren et al. 1997, Ren & Bode 2000). Several mechanisms are suggested for the pathogenesis of glucose-triggered cardiac dysfunction including prolonged action potential, impaired excitation–contraction coupling, loss of myofilament Ca2+ sensitivity, elevated oxidative stress, intracellular Ca2+ dysregulation and dampened insulin-like growth factor I (IGF-1) signaling (Cai & Kang 2001, Esberg & Ren 2003, Lagadic-Gossmann et al. 1996, Ren 2000, Duan et al. 2003). Most of these speculations have received convincing support from clinical and experimental studies using antioxidant, gene delivery and IGF-1 as therapeutic remedies for cardiovascular complications in diabetes (Goo et al. 1996, Ren et al. 1999a, Kajstura et al. 2001, Trost et al. 2002, Norby et al. 2002, 2004). Nevertheless, the precise cellular signaling mechanism behind glucose toxicity-induced cardiac mechanical dysfunction and the therapeutic value of IGF-1 against hyperglycemia remains poorly understood.

The mammalian target of rapamycin (mTOR), or FKBP-12 rapamycin-associated protein, is considered a critical player for cell signaling pathways from translational machinery, cell growth and metabolism to survival/ apoptosis in many cell types including ventricular myocytes. Phosphorylation of mTOR may be triggered by activation of its upstream molecules phosphatidylinositol 3 (PI-3) kinase and the serine/threonine kinase PKB/Akt (Hornberger et al. 2004, Oudit et al. 2004). Meanwhile, increased levels of nutrients, growth factors such as insulin, IGF-1 and amino acids may also turn on the mTOR signaling cascade through translational and phosphorylation mechanisms (Tremblay & Marette 2001, James & Zomerdijk 2004). However, direct contribution of mTOR to cardiac contractile function has not been reported. It was recently documented that altered signaling mechanism of mTOR, its upstream signaling molecules PI-3 kinase/Akt or its downstream activation of p70s6 kinase (p70s6k) is present in diabetic and insulin resistant conditions and may attribute to diabetes-associated metabolic and growth defects (Duan et al. 2003, Huisamen 2003, Um et al. 2004, Briaud et al. 2005). However, a direct link between elevated glucose levels and altered signaling of the PI-3 kinase/Akt, mTOR and p70s6k cascade has not been depicted in the hearts. Thus, the aim of our present study was to investigate the role of PI-3 kinase/Akt/mTOR/p70s6k in glucose toxicity-induced cardiac mechanical defects and IGF-1-elicited therapeutic effect against glucose toxicity. Possible contribution of the cyclosporin-inhibitable phosphatase calcineurin in IGF-1-induced cardiac contractile response was also tested since the calcineurin pathway is known to be involved in cardiac hypertrophic response of IGF-1 (Miyashita et al. 2001). We also evaluated glycogen synthase kinase-3β (GSK-3β), a ubiquitously expressed serine/threonine kinase and a key downstream signaling molecule of Akt serving as a negative regulatory target for Akt and p70s6k (Hardt & Sadoshima 2002). GSK-3β has been demonstrated to participate in a wide variety of biological effects including metabolism, gene transcription, protein translation, apoptosis, Ca2+ homeostasis and diastolic function (Hardt & Sadoshima 2002, Michael et al. 2004). We took advantage of a cardiac myocyte culture model developed in our lab, where diabetic cardiomyopathy may be phenotypically replicated in normal myocytes by culturing in a high glucose environment (Davidoff & Ren 1997, Esberg & Ren 2003, Ren et al. 2003). The abnormal excitation–contraction coupling is apparent in high glucose-treated myocytes reminiscent of those from in vivo diabetes (Davidoff & Ren 1997, Ren et al. 1997, 1999b, 2003).

Materials and Methods

Myocyte isolation

The experimental procedures used in this study were approved by the Institutional Animal Care and Use Committees at University of North Dakota (Grand Forks, ND, USA) and University of Wyoming (Laramie, WY, USA). All animal procedures were in accordance with the NIH animal care standards. In brief, adult male Sprague–Dawley rats weighing ~250 g were anesthetized with ketamine/xylazine (5:3, 1.32 mg/kg i.p.) before the hearts were rapidly removed and perfused (at 37 °C) with Krebs–Henseleit bicarbonate (KHB) buffer (mM: NaCl 118, KCl 4.7, CaCl2 1.25, MgSO4 1.2, KH2PO4 1.2, NaHCO3 25, N-[2-hydro-ethyl]-piperazine-N’-[2-ethanesulfonic acid] (HEPES) 10, glucose 11.1, pH 7.4). Hearts were subsequently perfused with Ca2+-free KHB containing 223 U/ml collagenase II (Worthington Biochemical Corp., Freehold, NJ, USA). After perfusion, the left ventricle was removed, minced and the cells were filtered through a nylon mesh (300 μm). Myocytes were washed with Ca2+-free KHB buffer to remove remnant enzyme, and extracellular Ca2+ was added incrementally and slowly back to 1.25 mM. Isolated myocytes were then maintained in a defined medium consisting of Medium 199 with Earle’s salts containing HEPES (25 mM) and NaHCO3 (25 mM), supplemented with albumin (2 mg/ml), l-carnitine (2 mM), creatine (5 mM), taurine (5 mM), insulin (100 nM), penicillin (100 U/ml), streptomycin (100 μg/ml), and gentamicin (5 μg/ml). This myocyte culture medium contained either normal (NG, 5.5 mM) or high (HG, 25.5 mM) glucose. The high extracellular glucose is comparable to serum glucose levels in diabetic rats (Ren & Bode 2000). A subset of each medium was also supplemented with either IGF-1 (10, 100 and 500 nM), the PI-3 kinase/Akt inhibitor LY294002 (10 μM), the mTOR inhibitor rapamycin (20 μM) and the calcineurin inhibitors cyclosporin A (5 μM) or tacrolimus (FK506, 10 mg/L), which functions through binding of FK506 binding protein 12 (FKBP12). DMSO was used as the solvent for some pharmacological inhibitors (e.g., LY294002) with a final concentration <0.2% in cell medium, which exerted no discernable effect on myocyte mechanics over the 8-h incubation period (data not shown). Our earlier studies indicated that the high glucose-induced myocyte contractile response was unlikely due to changes in extracellular osmotic pressure since replacement of glucose with equal molar (20 mM) D-mannitol exerted little effect on myocyte mechanics (Ren et al. 1997, 2003). The cells were maintained for 8 h at 37 °C in a 100% humidity and 5% CO2 incubator (Ren et al. 2003).

Cell shortening/relengthening

Mechanical properties of ventricular myocytes were assessed using a SoftEdge MyoCam system (IonOptix Corporation, Milton, MA, USA). In brief, left ventricular myocytes were placed in a Warner chamber mounted on the stage of an inverted microscope (Olympus, IX-70) and superfused (~1 ml/min at 25 °C) 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, 8 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 a computer monitor using an IonOptix MyoCam camera (IonOptix Corporation, Milton, MA, USA). IonOptix SoftEdge software was used to capture changes in cell length during shortening and relengthening (Ren et al. 2003).

Western blot analysis

Proteins from NG or HG-cultured ventricular myocytes with or without IGF-1 or selective pharmacological inhibitor treatment were extracted as described (Ren et al. 2003). Briefly, cardiac myocytes were lysed by sonication (3 × 5 s, 10 W) over ice in 0.5 ml RIPA lysis buffer containing 150 mM NaCl, 0.25 deoxycholic acid, 1% NP-40, 1 mM EDTA, 50 mM Tris–HCl, pH7.4, sodium orthovanadate 2 mM, and 1% protease inhibitor cocktail, and then by centrifugation (12 000 g, 10 min) to remove the precipitated material. Protein concentration was determined in the supernatant containing the soluble proteins using Bradford assay. The soluble proteins (50 μg/lane) were separated on 10% (Akt, pAkt, p70s6k, phosphor-p70s6k (pp70s6k), GSK-3β, phosphor-GSK-3β (pGSK-3β), phospholamban (PLB), phosphoserine of PLB, phosphothreonine of PLB) )or 7% (mTOR, phosphor-mTOR (pmTOR), sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA2a)) SDS-polyacrylamide gels in a minigel apparatus (Mini-PROTEAN II, Bio-Rad) and transferred to nitrocellulose membranes. The membranes were blocked with 5% milk in TBS-T, and then incubated with anti-Akt (1:1000), anti-pAkt (Thr308) (1:1000), anti-mTOR (1:1000), anti-pmTOR (Ser2448) (1:1000), anti-p70 S6 kinase (1:1000), anti-pp70 S6 kinase (1:1000), anti-GSK-3β (1:000) and phospho-GSK-3β (Ser9) (1:1000) antibodies (Cell Signaling Technology, Inc., Beverly, MA, USA), anti-SERCA2a antibody (1:2000, MA3–919, Affinity BioReagent, Denver, CO, USA), anti-PLB antibody (1:5000, 2D12 were kindly provided by Dr Steven Cala, Wayne State University School of Medicine, Detroit, MI, USA), and anti-phosphoserine of PLB (1:2000) and anti-phosphothreonine of PLB (1:2000) antibodies (Zymed Laboratory Inc. South San Francisco, CA, USA). The antigens were detected by the luminescence method (ECL Western blotting detection kit, Amersham) with peroxidase-linked anti-rabbit IgG (1:5000 dilution). After immunoblotting, the film was scanned and the intensity of immunoblot bands was detected with a Bio-Rad Calibrated Densitometer (Model: GS-800).

Statistics

Data are presented as means ± s.e.m. Statistical significance was ascertained by analysis of variance (ANOVA). When an overall significance was determined, a Dunnetts post hoc analysis was incorporated. P<0.05 was considered significant. Control groups (i.e., NG and HG cells) were always recorded on the same day as experimental groups (i.e., with and without drugs) in order to control for any potential inter-culture variability.

Results

IGF-1 protects against HG-induced prolonged relengthening

Culturing myocytes with HG, IGF-1 or pharmacological inhibitors had no overt effect on cell phenotype. Cell shape and presence of distinct striations were similar among all experimental groups. The resting cell length was comparable between NG and HG-cultured myocytes. However, incubation of IGF-1 appears to lengthen the resting cell length although such response was not consistent among the concentration range tested (Fig. 1A and 3A). As reported earlier (Davidoff & Ren 1997, Esberg & Ren 2003), myocytes maintained in HG medium exhibit reduced PS and ± dl/dt associated with prolonged TR90 and normal TPS compared with myocytes maintained in NG medium (Fig. 1–3). Interestingly, the reduction in PS, ± dL/dt and prolongation in TR90 in HG myocytes was ablated by 100 nM and 500 nM IGF-1 supplementations. Lower IGF-1 concentration (10 nM) did not affect the HG-induced mechanical defects. IGF-1 (100 nM) did not affect the mechanical properties in NG cells (Fig. 1–3). These results suggest that IGF-1 may effectively reconcile the HG environment induced cardiomyocyte mechanical defects simulating those in diabetes (Davidoff & Ren 1997, Ren & Bode 2000).

Inhibition of PI-3 kinase/Akt or mTOR but not calcineurin ablated IGF-1-induced protection against HG

To examine the potential signaling pathway(s) underscoring IGF-1-induced protection against glucose toxicity, NG and HG-cultured myocytes were co-incubated with selective blockers of PI-3 kinase/Akt, mTOR and calcineurin. Data shown in Figs 1, 2 and 3 indicated that the IGF-1-induced protection against HG-elicited mechanical defects was abolished by the PI-3 kinase/Akt inhibitor LY294002 and the mTOR inhibitor rapamycin but not the calcineurin inhibitors cyclosporine A or FK506. None of the inhibitors themselves affected myocyte mechanics with the exception that rapamycin prolonged TPS in HG myocytes (Figs 1–3. These data suggested involvement of PI-3 kinase/Akt and/or mTOR but unlikely calcineurin in IGF-1-induced protection against glucose toxicity-induced cardiomyocyte mechanical defects.

Western blot analysis of Akt, mTOR and p70s6k

IGF-1-induced cardiac contractile response has been shown to be mediated through post-receptor signaling pathways such as PI-3 kinase, the serine-theronine kinase Akt, mTOR and p70s6k (Ren 2000, Yamashita et al. 2001, Cui et al. 2003). This is supported by our mechanical data shown in Fig. 1 and Fig. 3. To further elucidate the role of Akt/mTOR/p70s6k under either NG or HG condition with or without IGF-1 addition, the protein expression of Akt, mTOR, p70s6k and activated forms of Akt, mTOR, p70s6k (pAkt, pmTOR, pp70s6k) were determined. Our immunostaining analysis revealed that while total Akt, total mTOR and total p70s6k levels were comparable among all cell groups (Fig. 4 and Fig. 5), the level of pAkt was both significantly and independently enhanced by HG and IGF-1. Paradoxically, IGF-1 significantly reduced HG-induced Akt phosphorylation. Inhibition of mTOR with rapamycin increased Akt phosphorylation in all groups tested (Fig. 4). Not surprisingly, IGF-1 stimulated the activation of mTOR shown as enhanced pmTOR in NG myocytes. HG significantly enhanced pmTOR and pp70s6k levels which may be attenuated by IGF-1 (Fig. 4 and Fig. 5). Activation of mTOR was abolished by rapamycin, as expected (Fig. 4).

Western blot analysis of GSK-3β, SERCA2a and PLB

Parallel to the mTOR/p70s6k pathway, the serine/ threonine kinase GSK-3β is another main downstream target for Akt activation (Hardt & Sadoshima 2002). We examined the expression of GSK-3β and phosphorylation of GSK-3β (phosph-GSK-3β), along with essential Ca2+-regulating proteins SERCA2a, PLB and serine/threonine phosphorylation of PLB. Our data suggested that while the total GSK-3β level was comparable among all cell groups, phosphorylation of GSK-3β (phospho-GSK-3β) was significantly reduced by HG which was ablated by IGF-1 (Fig. 5).

Protein expression of GSK-3β, SERCA2a, PLB and phosphorylated PLB was unaffected by HG, IGF-1 or rapamycin (except that rapamycin itself slightly but significantly suppressed serine phosphorylation of PLB) (Fig. 6).

Discussion

Results from our current study indicated that IGF-1-elicited protective effect against glucose toxicity-induced cardiac contractile dysfunctions. The beneficial effect of IGF-1 was mediated through a PI-3 kinase/Akt/mTOR/ p70s6k-dependent mechanism, rather than a calcineurin-dependent pathway. Our western blot analysis further revealed that both high glucose and IGF-1 stimulated phosphorylation of Akt and mTOR, although the high glucose-stimulated upregulation of pAkt and pmTOR may be paradoxically attenuated by IGF-1. Our gel electrophoresis data demonstrated that p70s6k and GSK-3β, both of which downstream signal molecules for PI-3 kinase/Akt, were activated and suppressed respectively, by high glucose. Consistent with observations from Akt and mTOR phosphorylation, co-incubation of IGF-1 with ventricular myocytes reconciled the high glucose-induced alteration in p70s6k and GSK-3β phosphorylation. Collectively, our data indicated a possible feedback mechanism of IGF-1 on high glucose-induced activation of Akt and mTOR, resulting in an ultimate protection from IGF-1 against high glucose-induced changes in phosphorylation of p70s6k and GSK-3β The unaltered expression of SERCA2a and PLB in high glucose-treated cells (with or without IGF-1 or rapamycin) indicated that the adverse effects of glucose toxicity on myocyte mechanics are unlikely mediated through translational modification of these key Ca2+ regulatory proteins.

Hyperglycemia is probably the most important predisposing factor in diabetes-related cardiovascular complications and directly contributes to the onset of cardiac dysfunction in diabetes (Sowers & Epstein 1995, Ren et al. 1997, 1999b). Data from our present study confirmed our earlier observations in that ventricular myocytes maintained in high glucose medium exhibited depressed cardiac contractility, reduced maximal velocity of contraction and relaxation and prolonged duration of relaxation, all of which are reminiscent of diabetic cardiomyopathy found in in vivo diabetes (Ren et al. 1997, Ren & Bode 2000). Prolonged relaxation and reduced cardiac contractility are hallmarks of diabetic cardiomyopathy and glucose toxicity-induced cardiomyocyte dysfunction (Davidoff & Ren 1997, Ren et al. 1999b, Esberg & Ren 2003). These glucose toxicity-induced cardiac mechanical defects are believed to be mediated through a number of cellular events including impaired intracellular Ca2+ handling, altered excitation–contraction coupling and dysregulated cellular enzyme activity or gene expression in cardiac myocytes (Gupta & Wittenberg 1993, Smogorzewski et al. 1998, Esberg & Ren 2003, Ren et al. 2003). Our present study revealed that IGF-1 may improve the high glucose-induced cardiac mechanical dysfunction, consistent with in vivo findings from our own lab as well as other labs, using diabetic rodent models (Kajstura et al. 2001, Norby et al. 2002, 2004). IGF-1 is known to facilitate glucose metabolism, lower insulin levels, improve insulin sensitivity and lipid profile (Ren et al. 1999a, Samarel 2002), suggesting both physiological and therapeutic roles of the peptide. Several scenarios have been speculated for IGF-1-induced cardiac protection against stress such as diabetes in addition to its well established action on glucose transport and lipid metabolism. For example, IGF-1 may regulate membrane ionic channels responsible for cardiac excitation–contraction coupling (Solem & Thomas 1998). IGF-1 may also elicit cardiac protection against diabetes involving attenuated p53 function, angiotensin II production and angiotensin receptor activation (Kajstura et al. 2001). Our studies suggested that IGF-1 may rescue the diabetes-induced reduction in SERCA protein abundance and impaired β-adrenergic responsiveness (Norby et al. 2002, 2004). In addition, diabetic tissues including hearts usually display IGF-1 resistance (Rodgers et al. 1995, Kelley et al. 1999, Ren et al. 1999a, Ren 2000, Duan et al. 2003), making glucose clearing by IGF-1 largely dependent on cross-reaction with the insulin receptor, which requires higher levels of IGF-1 agonist.

Perhaps the most significant finding from our results is that the IGF-1-offered cardiac protection against glucose toxicity is mediated via a PI-3 kinase/Akt/mTOR/ p70s6k-dependent rather than calcineurin-dependent pathway. Selective inhibitors of PI-3 kinase (LY294002) or mTOR (rapamycin) effectively ablated IGF-1-elicited cardiac protection against glucose-induced mechanical dysfunction in our cellular system. The PI-3 kinase/Akt/ mTOR/p70s6k pathway was recently emerged as a modulator of insulin (and IGF-1)-mediated glucose metabolism (Tremblay & Marette 2001, Tremblay et al. 2005) as well as cardiac growth and survival (Hornberger et al. 2004, Oudit et al. 2004). Our immunoblot observation indicated that IGF-1 directly stimulated Akt (in NG cells) and mTOR activation (in NG and HG cells), which is consistent with the previous findings (Yamashita et al. 2001, Cui et al. 2003, Ren et al. 2003). However, it is somewhat surprising that IGF-1 failed to stimulate phosphorylation of p70s6k, downstream signal molecule for Akt/mTOR. On the other hand, our results depicted that 8 h of high glucose treatment directly stimulates phosphorylation of Akt, mTOR and p70s6k. The impact of glucose on phosphorylation of Akt in the heart is somewhat controversial. Compromised Akt phosphorylation has been reported in both type 1 and type 2 diabetes as well as in cardiac myocytes exposed to a prolonged (24 h) high glucose environment (Huisamen 2003, Duan et al. 2003, Ren et al. 2003). Two scenarios may be considered for reduced Akt signaling in diabetes including ‘resistance’ to insulin-stimulated Akt activation and inhibition of Akt by elevated protein kinase C (PKC) activity in diabetes (Shiojima et al. 2002). However, recent evidence indicated that glucose-stimulated chronic Akt over-activation may contribute to ‘de-sensitization’ of Akt phosphorylation en route to insulin resistance (Ogihara et al. 2002). Over-phosphorylated Akt has been shown in cardiac tissues under both type 2 diabetes (Huisamen 2003) and hypertension (Ogihara et al. 2002). Hyper-insulinemia in insulin resistance and type 2 diabetes chronically activates Akt in the hearts, triggering a negative feedback loop to inhibit further Akt activation and excessive IRS-1 degradation (Haq et al. 2001). Thus, the activation status (acute vs chronic) of Akt is essential to cardiac function since acute Akt activation (such as via gene transfer) is known to regulate cell size, apoptosis, glucose metabolism, contraction and relaxation in cardiomyocytes (Latronico et al. 2004).

Probably the most puzzling findings from our study were that while both high glucose and IGF-1 stimulated phosphorylation of Akt and mTOR, IGF-1 itself significantly reduced the high glucose-induced activation of Akt, mTOR and p70s6k. Although we cannot provide a precise explanation of such findings at this time, several speculations may be made for this paradoxical phenomenon. First, it is possible that IGF-1-induced Akt phosphorylation is in the form of acute activation through IGF-1 or insulin receptor. On the other hand, high glucose-induced activation of Akt is in the form of chronic activation. While acute activation of PI-3 kinase/Akt has proven beneficial cardioprotective effects, the adverse consequences of chronic PI-3 kinase/Akt activation should be carefully elucidated. It was recently reported that chronic activation of Akt may lead to defective cardiac function (Nagoshi et al. 2005). Secondly, it is quite possible that activation of mTOR by IGF-1 may initiate a rapamycin-sensitive feedback phosphorylation of PI-3 kinase/Akt, which may diminish activated form Akt in high glucose environments (Fig. 7). This seems to be supported by the observation that rapamycin can overtly enhance the Akt phosphorylation in all groups. This is consistent with earlier reports that p70s6k may suppress the signaling through PI-3 kinase/Akt pathway, thus inhibition of p70s6k (through rapamycin) may boost Akt phosphorylation (Sarbassov et al. 2005). Inhibition of mTOR alone by rapamycin has also been demonstrated to stimulate Akt phosphorylation in a negative feedback manner (Tremblay et al. 2005). Thirdly, inhibition of mTOR has been shown to markedly potentiate the ability of insulin to increase phosphorylation of Akt at the levels of plasma membrane and cytosol (Tremblay et al. 2005). This again, is consistent with the higher Akt phosphorylation in the presence of rapamycin. It is thus possible that activation of mTOR, offered by the insulin analogue IGF-1 may reduce the Akt phosphorylation (stimulated by high glucose). Last but not least, mice deficient for p70s6k (S6K1), an effector of Akt/mTOR, were shown to be hypoinsulinaemic, glucose intolerant and protected against age- and diet-induced obesity with enhanced insulin sensitivity (Um et al. 2004). The enhanced p70s6k phosphorylation under high glucose environment is consistent with enhanced Akt/mTOR phosphorylation in high glucose-treated cardiomyocytes and underscored the likelihood of a protective mechanism for IGF-1 since IGF-1 effectively alleviated high glucose-induced p70s6k phosphorylation.

GSK-3β is a negative regulator of stress-induced cardiomyocyte hypertrophy and may serve to maintain normal cardiac growth, cell survival and contractile function (Michael et al. 2004). GSK-3β is normally active in un-stimulated cells and is inhibited when cells are exposed to either growth factors or the Wnt family of secreted glycoproteins. Our observation that the phosphorylation of GSK-3β was depressed in high glucose-treated myocytes is consistent with the notion that this signal molecule is negatively regulated by Akt and p70s6k (both of which were activated by HG in our experimental setting) (Hardt & Sadoshima 2002, Michael et al. 2004). De-regulation of GSK-3β through phosphorylation (in transgenic mice with wild-type GSK-3β) leads to severe systolic and diastolic dysfunction and progressive heart failure accompanied with down-regulation of SERCA2a (Michael et al. 2004). Whether IGF-1-induced reversal against high glucose-suppressed GSK-3β phosphorylation plays a role in IGF-1-offered cardiac mechanical protection is still elusive at this stage.

Taken together, our results indicate that the Akt/ mTOR/p70s6k pathway is an important modulator of the signals involved in glucose toxicity-induced mechanical dysfunction in cardiac myocytes and seems to play an essential role in IGF-1-offered cardiac protection against glucose toxicity. Further investigation is warranted to understand the precise mechanism underscoring the inter-relationship between the mTOR/p70s6k pathway and the PI-3 kinase/Akt pathway in the regulation of cardiomyocyte function and metabolism.

Figure 1
Figure 1

Contractile properties of left ventricular myocytes maintained for 8 h in medium containing either normal (NG, 5.5 mM) or high glucose (HG, 25.5 mM) supplemented with IGF-1 (10, 100 and 500 nM), the PI-3 kinase inhibitor LY294002 (10 μM) or the calcineurin inhibitor cyclosporin A (CsA, 5 μM). (A) Resting cell length (CL); (B) peak shortening (PS) normalized to CL; (C) maximal velocity of shortening (+ dl/dt); (D) maximal velocity of relengthening (− dl/dt); (E) time-to-PS (TPS); and (F) time-to-90% relengthening (TR90). Means ± s.e.m., n = 50–52 cells/group, *P<0.05 vs NG only group.

Citation: Journal of Endocrinology 186, 3; 10.1677/joe.1.06168

Figure 2
Figure 2

Contractile properties of left ventricular myocytes maintained for 8 h in NG or HG medium supplemented with IGF-1 (100 nM) or the calcineurin inhibitor FK506 (10 mg/l). (A) Peak shortening (PS) normalized to restign cell length (CL); (B) maximal velocity of shortening/relengthening (± dl/dt); (C) time-to-PS (TPS); and (D) time-to-90% relengthening (TR90). Means ± s.e.m., n = 60 cells/group, *P<0.05 vs NG group.

Citation: Journal of Endocrinology 186, 3; 10.1677/joe.1.06168

Figure 3
Figure 3

Contractile properties of left ventricular myocytes maintained for 8 h in NG or HG medium supplemented with IGF-1 (100 nM) or the mammalian target of rapamycin (mTOR) inhibitor rapamycin (20 μM). (A) Resting cell length (CL); (B) peak shortening (PS) normalized to resting cell length (CL); (C) maximal velocity of shortening (+ dl/dt); (D) maximal velocity of relengthening (− dl/dt); (E) time-to-PS (TPS); and (F) time-to-90% relengthening (TR90). Means ± s.e.m., n = 53–66 cells/group, *P<0.05 vs NG only group.

Citation: Journal of Endocrinology 186, 3; 10.1677/joe.1.06168

Figure 4
Figure 4

Protein abundance of Akt (A), pAkt (B), mammalian target of rapamycin (mTOR) (C) and p-mTOR (D) of myocytes maintained for 8 h in NG or HG medium supplemented with IGF-1 (100 nM) or the mTOR inhibitor rapamycin (20 μM). Insets are actual gel blots using anti-Akt, anti-pAkt, anti-mTOR and anti-p-mTOR antibodies with the same order of cell groups shown in the x-axis labels. Means ± s.e.m., n = 6–8 cultures, *P<0.05 vs NG only group, #P<0.05 vs HG only group.

Citation: Journal of Endocrinology 186, 3; 10.1677/joe.1.06168

Figure 5
Figure 5

Protein abundance of p70s6k (A), phospho-p70s6k (B), GSK-3β (C) and phospho-GSK-3β (D) of ventricular myocytes maintained for 8 h in NG or HG medium supplemented with IGF-1 (100 nM) or the mammalian target of rapamycin (mTOR) inhibitor rapamycin (20 μM). Insets are actual gel blots using anti-p70s6k, anti-phospho-p70s6k, anti-GSK-3β and anti-phospho-GSK-3β antibodies with the same order of groups shown in the x-axis labels. Means ± s.e.m., n = 4 cultures, *P<0.05 vs NG only group, #P<0.05 vs HG only group.

Citation: Journal of Endocrinology 186, 3; 10.1677/joe.1.06168

Figure 6
Figure 6

Protein abundance of SERCA2a (A), PLB (B), serine phosphorylation of PLB (C) and threonine phosphorylation of PLB (D) of myocytes maintained for 8 h in NG or HG medium supplemented with IGF-1 (100 nM) or the mammalian target of rapamycin (mTOR) inhibitor rapamycin (20 μM). Insets are actual gel blots using anti-SERCA2a, anti-PLB, anti-phosphoserine and anti-phosphothreonine antibodies with the same order of groups shown in the x-axis labels. Means ± s.e.m., n = 4 cultures, *P<0.05 vs NG only group.

Citation: Journal of Endocrinology 186, 3; 10.1677/joe.1.06168

Figure 7
Figure 7

Schematic figure depicting the model of signaling mechanisms for high glucose (solid lines) and IGF-1 (dotted lines) in cardiac myocytes. Note the feedback inhibition (solid vertical line to the left of PI-3 kinase/Akt) of IGF-1-induced mammalian target of rapamycin (mTOR) activation at the level of PI-3 kinase/Akt (triggered by high glucose).

Citation: Journal of Endocrinology 186, 3; 10.1677/joe.1.06168

*

(S-Y Li, C X Fang and N S Aberle II contributed equally to this work)

This work was supported by grants from the American Diabetes Association (07-RA-21), the American Heart Association Pacific Mountain Affiliate (#0355521Z), the National Institute of Aging (1R03 AG21324–01) and the University of Wyoming Faculty Grant-in-Aid to J R. IGF-1 was obtained from the National Hormone and Peptide Program, NIDDK and Dr Parlow (National Hormone and Peptide Program, NIDDK, NIH, Bethesda, MA, USA). The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.

References

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    • PubMed
    • Search Google Scholar
    • Export Citation
  • Cai L & Kang YJ 2001 Oxidative stress and diabetic cardiomyopathy: a brief review. Cardiovascular Toxicology 1 181–193.

  • Cui X, Zhang P, Deng W, Oesterreich S, Lu Y, Mills GB & Lee AV 2003 Insulin-like growth factor-I inhibits progesterone receptor expression in breast cancer cells via the phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin pathway: progesterone receptor as a potential indicator of growth factor activity in breast cancer. Molecular Endocrinology 17 575–588.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Davidoff AJ & Ren J 1997 Low insulin and high glucose induce abnormal relaxation in cultured adult rat ventricular myocytes. American Journal of Physiology – Heart and Circulatory Physiology 272 H159–H167.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Duan J, Zhang HY, Adkins SD, Ren BH, Norby FL, Zhang X, Benoit JN, Epstein PN & Ren J 2003 Impaired cardiac function and IGF-I response in myocytes from calmodulin-diabetic mice: role of Akt and RhoA. American Journal of Physiology – Endocrinology and Metabolism 284 E366–E376.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Esberg LB & Ren J 2003 Role of nitric oxide, tetrahydrobiopterin and peroxynitrite in glucose toxicity-associated contractile dysfunction in ventricular myocytes. Diabetologia 46 1419–1427.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Galderisi M, Anderson KM, Wilson PWF & Levy D 1991 Echocardiographic evidence for the existence of a distinct diabetic cardiomyopathy (the Framingham Heart Study). American Journal of Cardiology 68 85–89.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Goo AKY, Carson DS & Bjelajac A 1996 IGF-1: A new treatment option for non-insulin-dependent diabetes mellitus. Journal of Family Practice 42 612–618.

  • Gupta RK & Wittenberg BA 1993 19F nuclear magnetic resonance studies of free calcium in heart cells. Biophysical Journal 65 2547–2558.

  • Haq S, Choukroun G, Lim H, Tymitz KM, del Monte F, Gwathmey J, Grazette L, Michael A, Hajjar R, Force T & Molkentin JD 2001 Differential activation of signal transduction pathways in human hearts with hypertrophy versus advanced heart failure. Circulation 103 670–677.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Hardt SE & Sadoshima J 2002 Glycogen synthase kinase-3 beta: a novel regulator of cardiac hypertrophy and development. Circulation Research 90 1055–1063.

  • Hofmann PA, Menon V & Gannaway KF 1995 Effects of diabetes on isometric tension as a function of [Ca2+] and pH in rat skinned cardiac myocytes. American Journal of Physiology – Heart and Circulatory Physiology 269 H1656–H1663.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Hornberger TA, Stuppard R, Conley KE, Fedele MJ, Fiorotto ML, Chin ER & Esser KA 2004 Mechanical stimuli regulate rapamycin-sensitive signalling by a phosphoinositide 3-kinase-, protein kinase B- and growth factor-independent mechanism. Biochemistry Journal 380 795–804.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Huisamen B 2003 Protein kinase B in the diabetic heart. Molecular and Cellular Biochemistry 249 31–38.

  • James MJ & Zomerdijk JC 2004 Phosphatidylinositol 3-kinase and mTOR signaling pathways regulate RNA polymerase I transcription in response to IGF-1 and nutrients. Journal of Biological Chemistry 279 8911–8918.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Kajstura J, Fiordaliso F, Andreoli AM, Li B, Chimenti S, Medow MS, Limana F, Nadal-Ginard B, Leri A & Anversa P 2001 IGF-1 overexpression inhibits the development of diabetic cardiomyopathy and angiotensin II-mediated oxidative stress. Diabetes 50 1414–1424.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Kelley KM, Johnson TR, Ilan J & Moskowitz RW 1999 Glucose regulation of the IGF response system in chondrocytes: induction of an IGF-I-resistant state. American Journal of Physiology – Renal Physiology 276 R1164–R1171.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Lagadic-Gossmann DL, Buckler KJ, Le PK & Feuvray D 1996 Altered Ca2+ handling in ventricular myocytes isolated from diabetic rats. American Journal of Physiology – Heart and Circulatory Physiology 270 H1529–H1537.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Latronico MV, Costinean S, Lavitrano ML, Peschle C & Condorelli G 2004 Regulation of cell size and contractile function by AKT in cardiomyocytes. Annals of the New York Academy of Sciences 1015 250–260.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Michael A, Haq S, Chen X, Hsich E, Cui L, Walters B, Shao Z, Bhattacharya K, Kilter H, Huggins G, Andreucci M, Periasamy M, Solomon RN, Liao R, Patten R, Molkentin JD & Force T 2004 Glycogen synthase kinase-3 beta regulates growth, calcium homeostasis, and diastolic function in the heart. Journal of Biological Chemistry 279 21383–21393.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Miyashita T, Takeishi Y, Takahashi H, Kato S, Kubota I & Tomoike H 2001 Role of calcineurin in insulin-like growth factor-1-induced hypertrophy of cultured adult rat ventricular myocytes. Japanese Circulation Journal 65 815–819.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Nagoshi T, Matsui T, Aoyama T, Leri A, Anversa P, Li L, Ogawa W, del Monte F, Gwathmey J & Grazette L 2005 Phosphoinositide 3-kinase rescues the detrimental effects of chronic Akt activation in the heart during ischemia-reperfusion injury. Journal of Clinical Investigation 115 2128–2138.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Norby FL, Wold LE, Duan J, Hintz KK & Ren J 2002 IGF-I attenuates diabetes-induced cardiac contractile dysfunction in ventricular myocytes. American Journal of Physiology – Endocrinology and Metabolism 283 E658–E666.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Norby FL, Aberle NS II, Kajstura J, Anversa P & Ren J 2004 Transgenic overexpression of insulin-like growth factor I prevents streptozotocin-induced cardiac contractile dysfunction and beta-adrenergic response in ventricular myocytes. Journal of Endocrinology 180 175–182.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Ogihara T, Asano T, Ando K, Chiba Y, Sakoda H, Anai M, Shojima N, Ono H, Onishi Y, Fujishiro M, Katagiri H, Fukushima Y, Kikuchi M, Noguchi N, Aburatani H, Komuro I & Fujita T 2002 Angiotensin II-induced insulin resistance is associated with enhanced insulin signaling. Hypertension 40 872–879.

    • PubMed
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    • Search Google Scholar
    • Export Citation
  • Ren J 2000 Attenuated cardiac contractile responsiveness to insulin-like growth factor I in ventricular myocytes from biobreeding spontaneous diabetic rats. Cardiovascular Research 46 162–171.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Ren J & Bode AM 2000 Altered cardiac excitation–contraction coupling in ventricular myocytes from spontaneously diabetic BB rats. American Journal of Physiology – Heart and Circulatory Physiology 279 H238–H244.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Ren J & Davidoff AJ 1997 Diabetes rapidly induces contractile dysfunctions in isolated ventricular myocytes. American Journal of Physiology – Heart and Circulatory Physiology 272 H148–H158.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Ren J, Gintant GA, Miller RE & Davidoff AJ 1997 High extracellular glucose impairs cardiac E-C coupling in a glycosylation-dependent manner. American Journal of Physiology – Heart and Circulatory Physiology 273 H2876–H2883.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Ren J, Samson WK & Sowers JR 1999a Insulin-like growth factor I as a cardiac hormone: physiological and pathophysiological implications in heart disease. Journal of Molecular and Cellular Cardiology 31 2049–2061.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Ren J, Dominguez LJ, Sowers JR & Davidoff AJ 1999b Metformin but not glyburide prevents high glucose-induced abnormalities in relaxation and intracellular Ca2+ transients in adult rat ventricular myocytes. Diabetes 48 2059–2065.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Ren J, Duan J, Hintz KK & Ren BH 2003 High glucose induces cardiac insulin-like growth factor I resistance in ventricular myocytes: role of Akt and ERK activation. Cardiovascular Research 57 738–748.

    • PubMed
    • Search Google Scholar
    • Export Citation
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    • PubMed
    • Search Google Scholar
    • Export Citation
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    • Search Google Scholar
    • Export Citation
  • Shiojima I, Yefremashvili M, Luo Z, Kureishi Y, Takahashi A, Tao J, Rosenzweig A, Kahn CR, Abel ED & Walsh K 2002 Akt signaling mediates postnatal heart growth in response to insulin and nutritional status. Journal of Biological Chemistry 277 37670–37677.

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    • Search Google Scholar
    • Export Citation
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    • PubMed
    • Search Google Scholar
    • Export Citation
  • Tremblay F, Gagnon A, Veilleux A, Sorisky A & Marette A 2005 Activation of the mTOR pathway acutely inhibits insulin signaling to Akt and glucose transport in 3T3-L1 and human adipocytes. Endocrinology 146 1328–1337.

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  • Figure 1

    Contractile properties of left ventricular myocytes maintained for 8 h in medium containing either normal (NG, 5.5 mM) or high glucose (HG, 25.5 mM) supplemented with IGF-1 (10, 100 and 500 nM), the PI-3 kinase inhibitor LY294002 (10 μM) or the calcineurin inhibitor cyclosporin A (CsA, 5 μM). (A) Resting cell length (CL); (B) peak shortening (PS) normalized to CL; (C) maximal velocity of shortening (+ dl/dt); (D) maximal velocity of relengthening (− dl/dt); (E) time-to-PS (TPS); and (F) time-to-90% relengthening (TR90). Means ± s.e.m., n = 50–52 cells/group, *P<0.05 vs NG only group.

  • Figure 2

    Contractile properties of left ventricular myocytes maintained for 8 h in NG or HG medium supplemented with IGF-1 (100 nM) or the calcineurin inhibitor FK506 (10 mg/l). (A) Peak shortening (PS) normalized to restign cell length (CL); (B) maximal velocity of shortening/relengthening (± dl/dt); (C) time-to-PS (TPS); and (D) time-to-90% relengthening (TR90). Means ± s.e.m., n = 60 cells/group, *P<0.05 vs NG group.

  • Figure 3

    Contractile properties of left ventricular myocytes maintained for 8 h in NG or HG medium supplemented with IGF-1 (100 nM) or the mammalian target of rapamycin (mTOR) inhibitor rapamycin (20 μM). (A) Resting cell length (CL); (B) peak shortening (PS) normalized to resting cell length (CL); (C) maximal velocity of shortening (+ dl/dt); (D) maximal velocity of relengthening (− dl/dt); (E) time-to-PS (TPS); and (F) time-to-90% relengthening (TR90). Means ± s.e.m., n = 53–66 cells/group, *P<0.05 vs NG only group.

  • Figure 4

    Protein abundance of Akt (A), pAkt (B), mammalian target of rapamycin (mTOR) (C) and p-mTOR (D) of myocytes maintained for 8 h in NG or HG medium supplemented with IGF-1 (100 nM) or the mTOR inhibitor rapamycin (20 μM). Insets are actual gel blots using anti-Akt, anti-pAkt, anti-mTOR and anti-p-mTOR antibodies with the same order of cell groups shown in the x-axis labels. Means ± s.e.m., n = 6–8 cultures, *P<0.05 vs NG only group, #P<0.05 vs HG only group.

  • Figure 5

    Protein abundance of p70s6k (A), phospho-p70s6k (B), GSK-3β (C) and phospho-GSK-3β (D) of ventricular myocytes maintained for 8 h in NG or HG medium supplemented with IGF-1 (100 nM) or the mammalian target of rapamycin (mTOR) inhibitor rapamycin (20 μM). Insets are actual gel blots using anti-p70s6k, anti-phospho-p70s6k, anti-GSK-3β and anti-phospho-GSK-3β antibodies with the same order of groups shown in the x-axis labels. Means ± s.e.m., n = 4 cultures, *P<0.05 vs NG only group, #P<0.05 vs HG only group.

  • Figure 6

    Protein abundance of SERCA2a (A), PLB (B), serine phosphorylation of PLB (C) and threonine phosphorylation of PLB (D) of myocytes maintained for 8 h in NG or HG medium supplemented with IGF-1 (100 nM) or the mammalian target of rapamycin (mTOR) inhibitor rapamycin (20 μM). Insets are actual gel blots using anti-SERCA2a, anti-PLB, anti-phosphoserine and anti-phosphothreonine antibodies with the same order of groups shown in the x-axis labels. Means ± s.e.m., n = 4 cultures, *P<0.05 vs NG only group.

  • Figure 7

    Schematic figure depicting the model of signaling mechanisms for high glucose (solid lines) and IGF-1 (dotted lines) in cardiac myocytes. Note the feedback inhibition (solid vertical line to the left of PI-3 kinase/Akt) of IGF-1-induced mammalian target of rapamycin (mTOR) activation at the level of PI-3 kinase/Akt (triggered by high glucose).

  • Briaud I, Dickson LM, Lingohr MK, McCuaig JF, Lawrence JC & Rhodes CJ 2005 Insulin receptor substrate-2 proteasomal degradation mediated by a mammalian target of rapamycin (mTOR)-induced negative feedback down-regulates protein kinase B-mediated signaling pathway in beta-cells. Journal of Biological Chemistry 280 2282–2293.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Cai L & Kang YJ 2001 Oxidative stress and diabetic cardiomyopathy: a brief review. Cardiovascular Toxicology 1 181–193.

  • Cui X, Zhang P, Deng W, Oesterreich S, Lu Y, Mills GB & Lee AV 2003 Insulin-like growth factor-I inhibits progesterone receptor expression in breast cancer cells via the phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin pathway: progesterone receptor as a potential indicator of growth factor activity in breast cancer. Molecular Endocrinology 17 575–588.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Davidoff AJ & Ren J 1997 Low insulin and high glucose induce abnormal relaxation in cultured adult rat ventricular myocytes. American Journal of Physiology – Heart and Circulatory Physiology 272 H159–H167.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Duan J, Zhang HY, Adkins SD, Ren BH, Norby FL, Zhang X, Benoit JN, Epstein PN & Ren J 2003 Impaired cardiac function and IGF-I response in myocytes from calmodulin-diabetic mice: role of Akt and RhoA. American Journal of Physiology – Endocrinology and Metabolism 284 E366–E376.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Esberg LB & Ren J 2003 Role of nitric oxide, tetrahydrobiopterin and peroxynitrite in glucose toxicity-associated contractile dysfunction in ventricular myocytes. Diabetologia 46 1419–1427.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Galderisi M, Anderson KM, Wilson PWF & Levy D 1991 Echocardiographic evidence for the existence of a distinct diabetic cardiomyopathy (the Framingham Heart Study). American Journal of Cardiology 68 85–89.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Goo AKY, Carson DS & Bjelajac A 1996 IGF-1: A new treatment option for non-insulin-dependent diabetes mellitus. Journal of Family Practice 42 612–618.

  • Gupta RK & Wittenberg BA 1993 19F nuclear magnetic resonance studies of free calcium in heart cells. Biophysical Journal 65 2547–2558.

  • Haq S, Choukroun G, Lim H, Tymitz KM, del Monte F, Gwathmey J, Grazette L, Michael A, Hajjar R, Force T & Molkentin JD 2001 Differential activation of signal transduction pathways in human hearts with hypertrophy versus advanced heart failure. Circulation 103 670–677.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Hardt SE & Sadoshima J 2002 Glycogen synthase kinase-3 beta: a novel regulator of cardiac hypertrophy and development. Circulation Research 90 1055–1063.

  • Hofmann PA, Menon V & Gannaway KF 1995 Effects of diabetes on isometric tension as a function of [Ca2+] and pH in rat skinned cardiac myocytes. American Journal of Physiology – Heart and Circulatory Physiology 269 H1656–H1663.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Hornberger TA, Stuppard R, Conley KE, Fedele MJ, Fiorotto ML, Chin ER & Esser KA 2004 Mechanical stimuli regulate rapamycin-sensitive signalling by a phosphoinositide 3-kinase-, protein kinase B- and growth factor-independent mechanism. Biochemistry Journal 380 795–804.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Huisamen B 2003 Protein kinase B in the diabetic heart. Molecular and Cellular Biochemistry 249 31–38.

  • James MJ & Zomerdijk JC 2004 Phosphatidylinositol 3-kinase and mTOR signaling pathways regulate RNA polymerase I transcription in response to IGF-1 and nutrients. Journal of Biological Chemistry 279 8911–8918.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Kajstura J, Fiordaliso F, Andreoli AM, Li B, Chimenti S, Medow MS, Limana F, Nadal-Ginard B, Leri A & Anversa P 2001 IGF-1 overexpression inhibits the development of diabetic cardiomyopathy and angiotensin II-mediated oxidative stress. Diabetes 50 1414–1424.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Kelley KM, Johnson TR, Ilan J & Moskowitz RW 1999 Glucose regulation of the IGF response system in chondrocytes: induction of an IGF-I-resistant state. American Journal of Physiology – Renal Physiology 276 R1164–R1171.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Lagadic-Gossmann DL, Buckler KJ, Le PK & Feuvray D 1996 Altered Ca2+ handling in ventricular myocytes isolated from diabetic rats. American Journal of Physiology – Heart and Circulatory Physiology 270 H1529–H1537.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Latronico MV, Costinean S, Lavitrano ML, Peschle C & Condorelli G 2004 Regulation of cell size and contractile function by AKT in cardiomyocytes. Annals of the New York Academy of Sciences 1015 250–260.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Michael A, Haq S, Chen X, Hsich E, Cui L, Walters B, Shao Z, Bhattacharya K, Kilter H, Huggins G, Andreucci M, Periasamy M, Solomon RN, Liao R, Patten R, Molkentin JD & Force T 2004 Glycogen synthase kinase-3 beta regulates growth, calcium homeostasis, and diastolic function in the heart. Journal of Biological Chemistry 279 21383–21393.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Miyashita T, Takeishi Y, Takahashi H, Kato S, Kubota I & Tomoike H 2001 Role of calcineurin in insulin-like growth factor-1-induced hypertrophy of cultured adult rat ventricular myocytes. Japanese Circulation Journal 65 815–819.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Nagoshi T, Matsui T, Aoyama T, Leri A, Anversa P, Li L, Ogawa W, del Monte F, Gwathmey J & Grazette L 2005 Phosphoinositide 3-kinase rescues the detrimental effects of chronic Akt activation in the heart during ischemia-reperfusion injury. Journal of Clinical Investigation 115 2128–2138.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Norby FL, Wold LE, Duan J, Hintz KK & Ren J 2002 IGF-I attenuates diabetes-induced cardiac contractile dysfunction in ventricular myocytes. American Journal of Physiology – Endocrinology and Metabolism 283 E658–E666.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Norby FL, Aberle NS II, Kajstura J, Anversa P & Ren J 2004 Transgenic overexpression of insulin-like growth factor I prevents streptozotocin-induced cardiac contractile dysfunction and beta-adrenergic response in ventricular myocytes. Journal of Endocrinology 180 175–182.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Ogihara T, Asano T, Ando K, Chiba Y, Sakoda H, Anai M, Shojima N, Ono H, Onishi Y, Fujishiro M, Katagiri H, Fukushima Y, Kikuchi M, Noguchi N, Aburatani H, Komuro I & Fujita T 2002 Angiotensin II-induced insulin resistance is associated with enhanced insulin signaling. Hypertension 40 872–879.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Oudit GY, Sun H, Kerfant BG, Crackower MA, Penninger JM & Backx PH 2004 The role of phosphoinositide-3 kinase and PTEN in cardiovascular physiology and disease. Journal of Molecular and Cellular Cardiology 37 449–471.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Ren J 2000 Attenuated cardiac contractile responsiveness to insulin-like growth factor I in ventricular myocytes from biobreeding spontaneous diabetic rats. Cardiovascular Research 46 162–171.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Ren J & Bode AM 2000 Altered cardiac excitation–contraction coupling in ventricular myocytes from spontaneously diabetic BB rats. American Journal of Physiology – Heart and Circulatory Physiology 279 H238–H244.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Ren J & Davidoff AJ 1997 Diabetes rapidly induces contractile dysfunctions in isolated ventricular myocytes. American Journal of Physiology – Heart and Circulatory Physiology 272 H148–H158.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Ren J, Gintant GA, Miller RE & Davidoff AJ 1997 High extracellular glucose impairs cardiac E-C coupling in a glycosylation-dependent manner. American Journal of Physiology – Heart and Circulatory Physiology 273 H2876–H2883.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Ren J, Samson WK & Sowers JR 1999a Insulin-like growth factor I as a cardiac hormone: physiological and pathophysiological implications in heart disease. Journal of Molecular and Cellular Cardiology 31 2049–2061.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Ren J, Dominguez LJ, Sowers JR & Davidoff AJ 1999b Metformin but not glyburide prevents high glucose-induced abnormalities in relaxation and intracellular Ca2+ transients in adult rat ventricular myocytes. Diabetes 48 2059–2065.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Ren J, Duan J, Hintz KK & Ren BH 2003 High glucose induces cardiac insulin-like growth factor I resistance in ventricular myocytes: role of Akt and ERK activation. Cardiovascular Research 57 738–748.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Rodgers BD, Strack AM, Dallman MF, Hwa L & Nicoll CS 1995 Corticosterone regulation of insulin-like growth factor I, IGF-binding proteins, and growth in streptozotocin-induced diabetic rats. Diabetes 44 1420–1425.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Samarel AM 2002 IGF-1 overexpression rescues the failing heart. Circulation Research 90 631–633.

  • Sarbassov DD, Guertin DA, Ali SM & Sabatini DM 2005 Phosphorylation and regulation of Akt/PKB by the rictor-mTOR complex. Science 307 1098–1101.

  • Schaffer SW, Ballard-Croft C, Boerth S & Allo AN 1997 Mechanisms underlying depressed Na+/Ca2+ exchanger activity in the diabetic heart. Cardiovascular Research 34 129–136.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Shiojima I, Yefremashvili M, Luo Z, Kureishi Y, Takahashi A, Tao J, Rosenzweig A, Kahn CR, Abel ED & Walsh K 2002 Akt signaling mediates postnatal heart growth in response to insulin and nutritional status. Journal of Biological Chemistry 277 37670–37677.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Smogorzewski M, Galfayan V & Massry SG 1998 High glucose concentration causes a rise in [Ca2+]i of cardiac myocytes. Kidney International 53 1237–1243.

  • Solem ML & Thomas AP 1998 Modulation of cardiac Ca2+ channels by IGF-1. Biochemical and Biophysical Research Communications 252 151–155.

  • Sowers JR & Epstein M 1995 Diabetes mellitus and associated hypertension, vascular disease and nephropathy: an update. Hypertension 26 869–879.

  • Tremblay F & Marette A 2001 Amino acid and insulin signaling via the mTOR/p70 S6 kinase pathway. A negative feedback mechanism leading to insulin resistance in skeletal muscle cells. Journal of Biological Chemistry 276 38052–38060.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • Tremblay F, Gagnon A, Veilleux A, Sorisky A & Marette A 2005 Activation of the mTOR pathway acutely inhibits insulin signaling to Akt and glucose transport in 3T3-L1 and human adipocytes. Endocrinology 146 1328–1337.

    • PubMed
    • Search Google Scholar
    • Export Citation
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