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(Circulation Research. 2005;96:180.)
© 2005 American Heart Association, Inc.

Cellular Biology

Akt Mediates the Cross-Talk Between ß-Adrenergic and Insulin Receptors in Neonatal Cardiomyocytes

Carmine Morisco, Gerolama Condorelli, Valentina Trimarco, Alessandro Bellis, Chiara Marrone, Gianluigi Condorelli, Junichi Sadoshima, Bruno Trimarco

From the Dipartimento di Medicina Clinica, Scienze Cardiovascolari ed Immunologiche (C. Morisco, V.T., A.B., C. Marrone, B.T.), Dipartimento di Biologia e Patologia Cellulare e Molecolare (Ge.C.), Università Federico II, Napoli, Italy; San Raffaele Biomedical Science Park of Rome (Gi.C.), Italy; and the Department of Cell Biology and Molecular Medicine (J.S.), University of Medicine and Dentistry of New Jersey, Newark.

Correspondence to Bruno Trimarco, MD, Dipartimento di Medicina Clinica, Scienze Cardiovascolari ed Immunologiche, Università Federico II, Napoli, Via S. Pansini n. 5, 80131 Napoli, Italy. E-mail trimarco{at}unina.it

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Upregulation of the sympathetic nervous system plays a key role in the pathogenesis of insulin resistance. Although the heart is a target organ of insulin, few studies have examined the mechanisms by which ß-adrenergic stimulation affects insulin sensitivity in cardiac muscle. In this study, we explored the molecular mechanisms involved in the regulation of the cross-talk between ß adrenergic and insulin receptors in neonatal rat cardiomyocytes and in transgenic mice with cardiac overexpression of a constitutively active mutant of Akt (E40K Tg). The results of this study show that ß-adrenergic receptor stimulation has a biphasic effect on insulin-stimulated glucose uptake. Short-term stimulation induces an additive effect on insulin-induced glucose uptake, and this effect is mediated by phosphorylation of Akt in threonine 308 through PKA/Ca²⁺-dependent and PI3K-independent pathway, whereas insulin-evoked threonine phosphorylation of Akt is exclusively PI3K-dependent. On the other hand, long-term stimulation of ß-adrenergic receptors inhibits both insulin-stimulated glucose uptake and insulin-induced autophosphorylation of the insulin receptor, and at the same time promotes threonine phosphorylation of the insulin receptor. This is mediated by serine 473 phosphorylation of Akt through PKA/Ca²⁺ and PI3K-dependent pathways. Under basal conditions, E40K Tg mice show increased levels of threonine phosphorylation of the ß subunit of the insulin receptor and blunted tyrosine autophosphorylation of the ß-subunit of the insulin receptor after insulin stimulation. These results indicate that, in cardiomyocytes, ß-adrenergic receptor stimulation impairs insulin signaling transduction machinery through an Akt-dependent pathway, suggesting that Akt is critically involved in the regulation of insulin sensitivity.

Key Words: glucose uptake • isoproterenol • insulin resistance • protein kinase A • L-type Ca²⁺ channel

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Insulin resistance plays an important role in the pathogenesis of diabetes,^1,2 obesity,³ and hypertension,^4,5 and is also a common feature of heart failure.^6,7 Dysregulation of the sympathetic nervous system has been reported in obesity,⁸ contributes to the etiology of hypertension,^9,10 leads to an adverse prognosis in heart failure,¹¹ and is involved in the pathogenesis of insulin resistance.^7,9

The term "insulin resistance" refers to the action of insulin on glucose homeostasis, and it has been demonstrated that skeletal muscle¹² and adipose tissue¹³ are the organs that mainly participate in the development of insulin resistance. Cardiac muscle is also a target of insulin,¹⁴ and impairment of insulin-stimulated cardiac glucose uptake has been described in animal models of diabetes,¹⁵ obesity,¹⁶ and hypertension.^17,18 However, few studies have investigated the cross-talk between ß-adrenergic and insulin signaling in the heart.

Binding of insulin to its receptor activates the tyrosine kinase activity of the ß-subunit of the receptor,¹⁹ leading to autophosphorylation, as well as tyrosine phosphorylation of several insulin receptor (IR) substrates. These, in turn, interact with phosphatidylinositol 3-kinase (PI3K). Activation of PI3K stimulates the downstream effector Akt, a serine/threonine kinase, which induces glucose uptake through the translocation of the glucose transporter GLUT4 to the plasma membrane.²⁰ Abnormalities of insulin signaling account for insulin resistance. Several mechanisms have been described as being responsible for the inhibition of insulin-stimulated tyrosine phosphorylation of IR, including proteasome-mediated degradation,²¹ phosphatase-mediated dephosphorylation,²² and kinase-mediated serine/threonine phosphorylation.²³

The cross-talk between ß-adrenergic and insulin signaling has been investigated by Klein et al²⁴ that have demonstrated in cultured adipocytes that insulin-induced glucose uptake and tyrosine phosphorylation of the IR were inhibited by ß₃-adrenergic receptor (AR) stimulation.

Myocardial ßARs consist of ß₁ and ß₂ subtypes,²⁵ although recent evidence suggests that a small population of the ß₃ subtype also exists in the heart.²⁶ Ligand binding to different ßAR subtypes activates different signaling mechanisms.²⁷ Therefore, depending on the distribution of ßAR subtypes, different cell types have specific molecular mechanisms involved in the regulation of the cross-talk between the insulin and ß-adrenergic systems. Because Akt is a serine/threonine kinase, which can be activated by ßAR,²⁸ it is reasonable to hypothesize that, after ßAR stimulation, Akt phosphorylates the ß-subunit of IR.

In this study, we explored in cardiomyocytes and in transgenic mice the mechanisms involved in the regulation of the cross-talk between the ß-adrenergic and insulin systems. We examined (1) the effects of short- and long-term stimulation of ßAR on insulin-induced glucose uptake, (2) whether Akt participates in the cross-talk between insulin and ßAR, and (3) whether cardiac overexpression of a constitutively active mutant of Akt impairs insulin signaling in vivo.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Primary Cultures of Neonatal Rat Ventricular Cardiomyocytes
Primary cultures of neonatal cardiomyocytes were prepared as we have previously described.²⁹

Glucose Uptake Assays
Cardiomyocytes were grown in 12-well plates. 2-Deoxyglucose (2DG) uptake was determined by the method of Moyers et al³⁰ with few modifications (for details, see the online data supplement available at http://circres.ahajournals.org).

Immunoblotting
Cardiomyocytes were grown in 6-well plates. At the end of the stimulation period, the medium was removed, the cells washed twice with ice-cold Ca²⁺/Mg²⁺-free Dulbecco PBS, and lysed with 100 µL of ice-cold lysis buffer A (see online data supplement). Phosphorylation of Akt was detected with anti–phospho-Akt (Ser473) (Cell Signaling Technology) and with anti–phospho-Akt (Thr308) (Cell Signaling Technology), Akt was determined with anti-Akt antibody (Cell Signaling Technology). Horseradish peroxidase-conjugated (Cell Signaling Technology) antibody was used as secondary antibody. The bound secondary antibody was detected by enhanced chemiluminescence (Amersham Pharmacia Biotec).

Immunoprecipitation and Detection of Phospho-Akt Substrate
Cardiomyocytes were grown in 60-mm dishes. At the end of the stimulation period, the medium was removed, the cells washed twice with ice-cold Ca²⁺/Mg²⁺-free Dulbecco PBS and lysed with 1 mL of ice-cold lysis buffer B (see online data supplement). IR was immunoprecipitated with anti-IR ß-subunit (Santa Cruz Biotechnology, Inc) and with protein A/G-Sepharose slurry (Santa Cruz Biotechnology, Inc). Immunoprecipitates were subjected to SDS-PAGE, transferred to a polyvinylidene diflouride membrane, and immunoblotted with anti-IR ß-subunit (Santa Cruz Biotechnology, Inc), anti-phosphotyrosine (Cell Signaling Technology), and anti-phosphothreonine (Cell Signaling Technology) antibodies, and with anti–phospho-Akt substrate antibody (Cell Signaling Technology).

Akt Kinase Activity Assay
Kinase activity of Akt was measured by the immune complex kinase assay as we have previously described.²⁸

Adenovirus Transduction
Cardiomyocytes were infected with adenoviruses harboring dominant-negative (Ad5 CMV Akt [K179M]) and constitutively active (Akt, E40K) Akt. Adenovirus harboring lacZ (Ad5 · CMV-ß-galactosidase) was used as a control. The method of adenovirus infection has been previously described.³¹

In Vivo Studies
Three-month-old transgenic (Tg) mice with cardiac specific overexpression of constitutively active mutant (E40K) of Akt³² and wild-type controls were studied (for details, see online data supplement)

Statistics
Data are given as mean±SEM. Statistical analyses were performed using analysis of variance. The posttest comparison was performed by the method of Tukey. Significance was accepted at P<0.05 levels.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Characteristics of Insulin- and ISO-Induced 2DG Uptake in Neonatal Cardiomyocytes
We first assessed the dose-response relationship and the time course of insulin-induced 2DG uptake. Thirty minutes of insulin stimulation dose-dependently increased 2DG uptake. A significant increase in 2DG uptake was observed at 1 nmol/L, and it reached a plateau at 100 nmol/L, with an EC₅₀ of 3.25 nmol/L (Figure 1A). Insulin and isoproterenol (ISO), a ßAR agonist, both induced 2DG uptake, peaking after 30 (Figure 1B) and 10 (Figure 1C) minutes, respectively.

View larger version (24K): [in this window] [in a new window]   Figure 1. Insulin and ßAR stimulation dose- and time-dependently increase 2-deoxyglucose uptake. A, Cardiomyocytes were stimulated for 30 minutes with graded doses of insulin ranging from 10 pmol/L to 10 µmol/L, and the rate of 2-deoxyglucose (2DG) uptake was determined. Graphics show the mean±SEM of 3 independent experiments. B, Cardiomyocytes were stimulated with insulin (Ins, 100 nmol/L) for the times indicated, and the rate of 2DG uptake was determined. C, Cardiomyocytes were stimulated with isoproterenol (ISO, 10 µmol/L), and the rate of 2DG uptake was determined. Values of 2DG uptake (cpm) were adjusted by the protein content (µg). B and C, Mean±SEM of 5 independent experiments.

Short-Term ß-Adrenergic Stimulation Increases Insulin-Induced 2DG Uptake Through a PKA/Ca²⁺-Dependent Pathway
Next, we evaluated the effects of short-term ßAR stimulation on insulin-induced 2DG uptake. Ten minutes of stimulation with ISO enhanced 2DG uptake by 36±2% (P<0.05 versus control). However, the increases in 2DG uptake were not as large as that obtained with insulin (100 nmol/L), which induced an increase of 122±12%. Pretreatment of cardiomyocytes with ISO for 10 minutes had an additive effect on insulin-induced 2DG uptake (164±9% versus control), suggesting that insulin and ISO use two different mechanisms to stimulate 2DG uptake (Figure 2A). Therefore, we examined the mechanisms that account for ISO and insulin-induced 2DG uptake. ßARs act through the cAMP/PKA/L-type Ca²⁺ channel–dependent pathway,³³ whereas IR uses mainly the IRS1–2/PI3K–dependent pathway. Inhibition of PKA, and kinase Ca²⁺-calmodulin–dependent, obtained with H89 and KN93, respectively, as well as the Ca²⁺ antagonist, nifedipine, blocked short-term ISO-induced 2DG uptake (Figure 2B), whereas PI3K inhibition by wortmanin did not. In contrast, insulin-induced 2DG uptake was found to be PI3K dependent (Figure 2C). Because the effects of ßAR simulation on 2DG uptake seemed to be mediated by a Ca²⁺-dependent pathway, we investigated the role of Ca²⁺ in the regulation of 2DG uptake. Treatment of cardiomyocytes with Ca²⁺ ionophore A23187 increased 2DG uptake by 37±3% (P<0.05 versus control), and enhanced the effect of insulin to the same extent as that of ISO (Figure 2D).

View larger version (44K): [in this window] [in a new window]   Figure 2. Insulin and ßAR stimulation induce 2-deoxyglucose uptake by two different mechanisms. A, Cardiomyocytes were stimulated for 30 minutes with Ins (100 nmol/L), for 10 minutes with ISO (10 µmol/L), and with ISO+Ins (10 and 30 minutes, respectively), and the rate of 2DG uptake was determined. B, Cardiomyocytes were stimulated with ISO for 10 minutes in the absence or presence of pretreatment with H89 (10 µmol/L, 60 minutes), KN93 (0.2 µmol/L, 30 minutes), nifedipine (0.1 µmol/L, 60 minutes), and wortmanin (10 nmol/L, 30 minutes), and the rate of 2DG uptake was determined. C, Cardiomyocytes were stimulated with Ins for 30 minutes in the absence or presence of pretreatment with H89, KN93, nifedipine, and wortmanin, and the rate of 2DG uptake was determined. D, Cardiomyocytes were stimulated for 30 minutes with Ins, for 10 minutes with ISO, with ISO+Ins, and with Ca2+ ionophore A23187 (1 µmol/L, 6 hours) with or without Ins, and the rate of 2DG uptake was determined. Values of 2DG uptake (cpm) were adjusted by the protein content of the dish (µg). Graphs show the mean±SEM of 5 independent experiments.

These data suggest that insulin and ßAR stimulation induce 2DG uptake by PI3K-dependent and PKA/Ca²⁺-dependent pathways, respectively.

ßAR Stimulation Induces 2DG Uptake Through an Akt-Dependent Pathway
Stimulation of ßAR activates Akt,²⁸ which is the key molecule involved in the regulation of glucose uptake.²⁰ Therefore, we asked if Akt is involved in ISO-induced glucose uptake. Cardiomyocytes were infected with adenovirus harboring lacZ or dominant-negative Akt (DN Akt) (Figure 3A). Immune complex kinase assays showed that 10 minutes of ISO stimulation increased kinase activity of Akt, whereas overexpression of DN Akt inhibited this response (Figure 3B). Furthermore, overexpression of DN Akt inhibited ISO-induced 2DG uptake (Figure 3C), indicating that Akt is required for ISO-induced 2DG uptake.

View larger version (30K): [in this window] [in a new window]   Figure 3. ßAR stimulation induces 2-deoxyglucose uptake through an Akt-dependent pathway. A, Cardiomyocytes were infected with the indicated doses of recombinant adenovirus harboring dominant-negative Akt (DN-Akt) or control adenovirus harboring Lac Z, and cultured under serum-free conditions for 48 hours. Top, Immunoblot with anti-Akt antibody shows the expression level of transgenes in infected myocytes. Bottom, Immunoblot with anti-actin antibody shows the expression level of actin in infected myocytes. B, Cardiomyocytes were infected with LacZ and DN-Akt, cultured for 48 hours, and then stimulated with ISO (10 µmol/L) for 10 minutes. Kinase activity of Akt was determined. Similar results were obtained in four other experiments. C, Cardiomyocytes were infected with LacZ and DN-Akt, cultured under serum-free conditions for 48 hours, and then stimulated with ISO for 10 minutes (10 µmol/L). Rate of 2DG uptake was determined. Values of 2DG uptake (cpm) were adjusted by the protein content of the dish (µg). Graph shows the mean±SEM of 5 independent experiments.

Short-Term ßAR Stimulation and Insulin Phosphorylate Akt in Thr308 Through Different Pathways
ISO stimulation of cardiomyocytes induces Akt phosphorylation both in threonine (Thr) 308 and in serine (Ser) 473 with different time-course. In particular, ISO-induced Thr phosphorylation was detectable after 1 minute, peaked after 10 minutes, and then progressively decreased, whereas Ser phosphorylation started after 10 minutes, peaked after 60 minutes, and then decreased (Figure 4A). Considering the time course of both ISO-induced 2DG uptake and Akt Thr and Ser phosphorylation, it is likely that ISO-stimulated 2DG uptake was mediated by Thr phosphorylation of Akt. Therefore, we explored the pathway involved in ISO-induced phosphorylation of Akt in Thr308. Treatment of cardiomyocytes with H89, KN93, and nifedipine inhibited, whereas wortmanin did not affect, ISO-induced phosphorylation of Akt in Thr308 (Figure 4B). In contrast, insulin-induced phosphorylation of Akt in Thr308 was detectable after 5 minutes of stimulation (Figure 4C) and was inhibited in the presence of wortmanin (Figure 4D).

View larger version (43K): [in this window] [in a new window]   Figure 4. Short-term ßAR stimulation and insulin phosphorylate Akt in Thr308 through different pathways. A, Cardiomyocytes were stimulated with ISO (10 µmol/L). Phosphorylation of Akt in threonine and in serine was detected by immunoblotting analyses with anti–phospho-Thr308 Akt (Top), and anti–phospho-Ser473 Akt (bottom) antibodies. B, Cardiomyocytes were preincubated with or without H89, KN93, nifedipine, and wortmanin, and then stimulated with ISO for 10 minutes. Phosphorylation of Akt in threonine was assessed by immunoblotting analysis. C, Cardiomyocytes were stimulated with Ins (100 nmol/L) for the indicated durations. Phosphorylation of Akt in threonine was assessed by immunoblotting analysis. D, Cardiomyocytes were preincubated with or without H89, KN93, nifedipine, and wortmanin and then stimulated with Ins for 30 minutes. Phosphorylation of Akt in threonine was assessed by immunoblotting analysis. All experiments were performed in quadruplicate.

It is possible that short-term ßAR stimulation and insulin use two different pathways to phosphorylate Akt in Thr308 and consequently have an additive effect on 2DG uptake.

Long-Term ßAR Stimulation Inhibits Insulin-Induced 2DG Uptake by Interfering With Tyrosine Phosphorylation of IR
We next determined whether or not long-term ISO stimulation interferes with insulin-induced 2DG uptake. For this purpose, cardiomyocytes were incubated with ISO at different time points ranging from 10 to 120 minutes, then were stimulated with insulin for 30 minutes. Insulin stimulation enhanced 2DG uptake detected at 10 and 30 minutes of ISO pretreatment compared with insulin alone. Thereafter, insulin-induced 2DG uptake started to decrease and was completely inhibited after 120 minutes of ISO exposure (Figure 5A), indicating that long-term ßAR stimulation is able to induce insulin resistance.

View larger version (29K): [in this window] [in a new window]   Figure 5. Long-term ßAR stimulation inhibits insulin-induced 2-deoxyglucose uptake by interfering with tyrosine phosphorylation of the insulin receptor. A, Cardiomyocytes were preincubated with ISO for the times indicated, and stimulated with Ins for 30 minutes, and the rate of 2DG uptake was determined. Values of 2DG uptake (cpm) were adjusted by the protein content of the dish (µg). Graph shows the mean±SEM of 5 independent experiments. B, Cardiomyocytes were preincubated with ISO (10 µmol/L) for the times indicated and were stimulated with Ins (100 nmol/L) for 5 minutes. Protein lysates were subjected to immunoprecipitation with antibodies against insulin receptor (IR) ß-subunit, followed by immunoblotting using anti-phosphotyrosine antibody. C, Cardiomyocytes were stimulated with ISO at a concentration of 10 µmol/L for the times indicated. Protein lysates were subjected to immunoprecipitation with antibodies against IR ß-subunit, followed by immunoblotting using anti-phosphothreonine antibody. All experiments were performed in quadruplicate.

Next, we investigated whether or not long-term ISO stimulation interferes with insulin-induced tyrosine autophosphorylation of the IR. Cardiomyocytes were treated with ISO at different time points, and then stimulated with insulin for 5 minutes. As expected, insulin stimulated tyrosine autophosphorylation of the ß-subunit of its receptor. Interestingly, insulin-induced tyrosine phosphorylation of the ß-subunit was time-dependently inhibited by ISO pretreatment and was almost totally abolished after 120 minutes of ßAR stimulation (Figure 5B).

Because insulin-stimulated tyrosine autophosphorylation of its receptor can be inhibited by either phosphatase-mediated dephosphorylation or kinase-mediated serine/threonine phosphorylation, we asked which mechanism accounts for the inhibition of tyrosine phosphorylation of IR induced by long-term ISO stimulation. Sixty minutes of pretreatment with 50 µmol/L orthovanadate, an inhibitor of tyrosine phosphatases, did not affect the inhibitory effects of long-term ISO stimulation on insulin-induced tyrosine autophosphorylation of the IR (data no shown). By contrast, 120 minutes of ISO stimulation induced threonine phosphorylation of the ß-subunit of the IR (Figure 5C). These results suggest that long-term ISO stimulation induces threonine phosphorylation of the ß-subunit of IR, which in turn inhibits insulin-induced tyrosine autophosphorylation of the receptor.

Akt Mediates ß-Adrenergic Receptor Stimulation-Induced Threonine Phosphorylation of the ß-Subunit of IR
Next, we asked if ISO-induced threonine phosphorylation of IR is mediated by Akt. First, we explored whether or not IR is a substrate for Akt. Cardiomyocytes were stimulated with ISO at different time points, then the lysates were immunoprecipitated with an antibody against the ß-subunit of IR and blotted with an antibody that binds peptides/proteins that contain phospho-Thr/Ser preceded by Arg/Lys at positions -5 and -3, which are the consensus motifs recognized by Akt. ISO stimulation time-dependently determined the recognition of the ß-subunit of IR by anti Akt substrate antibody starting from 60 minutes (Figure 6A). This suggests that ISO stimulation activates Akt, which in turn, recognizes IR as a substrate. Interestingly, the time-course of Akt phosphorylation in Ser induced by ISO (Figure 4A) suggests that the phosphorylation in Ser mediates the interaction of Akt with IR. Therefore, we determined whether or not inhibition of Ser phosphorylation of Akt restores insulin-induced tyrosine phosphorylation of IR after long-term ISO stimulation. ISO-induced Ser phosphorylation of Akt was inhibited by H89, KN93, nifedipine, and wortmanin, indicating that it is a PKA/Ca²⁺- and PI3K-dependent phenomenon (Figure 6B). Coincidentally, inhibition of both PKA/Ca²⁺-dependent and PI3K-dependent pathways (Figure 6C) restored insulin-induced tyrosine phosphorylation of IR after long-term ßAR stimulation. To explore whether Akt plays a mechanistic role in the regulation of tyrosine phosphorylation of IR, DN Akt, and E40K-Akt mutants were overexpressed in cardiac myocytes. DN Akt restored insulin-induced tyrosine phosphorylation of IR after long-term ßAR stimulation, and E40K-Akt blunted insulin-induced tyrosine phosphorylation of IR (Figure 6D).

View larger version (45K): [in this window] [in a new window]   Figure 6. Akt mediates ßAR stimulation–induced threonine phosphorylation of the ß-subunit of the insulin receptor. A, Cardiomyocytes were stimulated with ISO (10 µmol/L) for the times indicated. Protein lysates were subjected to immunoprecipitation with antibodies against insulin receptor (IR) ß-subunit, followed by immunoblotting using phospho-Akt substrate antibody (top). Relative quantities of the IR determined by reblot in this immunoprecipitation experiment are also shown (bottom). B, Cardiomyocytes were stimulated with ISO (10 µmol/L, 60 minutes) in the absence or presence of H89, KN93, nifedipine and wortmanin. Phosphorylation of Akt in serine was detected by immunoblotting analyses. C, Cardiomyocytes were preincubated with or without H89, KN93, nifedipine, and wortmanin then were stimulated with ISO at a concentration of 10 µmol/L for 120 minutes followed by 5 minutes of Ins (100 nmol/L) stimulation. Protein lysates were subjected to immunoprecipitation with antibodies against IR ß-subunit, followed by immunoblotting using anti-phosphotyrosine antibody. D, Cardiomyocytes were infected with adenoviruses harboring LacZ, DN-Akt, or E40K Akt cultured under serum-free conditions for 48 hours, and then stimulated with ISO for 120 minutes (10 µmol/L) followed by 5 minutes of Ins stimulation, or with Ins alone. Protein lysates were subjected to immunoprecipitation with antibodies against IR ß-subunit, followed by immunoblotting using anti-phosphotyrosine antibody. All experiments were performed in quadruplicate.

These data suggest that after long-term ßAR stimulation, Ser phosphorylation of IR is mediated by Akt through PKA/Ca²⁺- and PI3K-dependent pathways.

Cardiac Overexpression of Akt Impairs Insulin Signaling In Vivo
To verify whether activation of Akt impairs insulin signaling in vivo, we examined the hearts of Tg mice with cardiac overexpression of a constitutively active mutant of Akt (E40K) and wild-type controls for insulin-induced phosphorylation of IR. In WT mice, as expected, insulin induced tyrosine phosphorylation of its receptor’s ß-subunit. This phenomenon was blunted in E40K Tg mice (Figure 7A).

View larger version (37K): [in this window] [in a new window]   Figure 7. Cardiac overexpression of a constitutively active mutant of Akt interferes with insulin-induced tyrosine phosphorylation of the insulin receptor. A, E40K Tg mice and wild-type controls (WT) were injected with saline (Vehic) or Ins (10 UI/Kg of body weight). After 5 minutes, the hearts were removed, and insulin-induced insulin receptor tyrosine autophosphorylation was determined by immunoprecipitation against the ß-subunit of the IR followed by immunoblotting using an anti–phosphotyrosine antibody. B, E40K Tg and WT mice were infused with saline or ISO (0.05 µg/Kg of body weight) for 30 minutes. Hearts were then removed, and insulin receptor threonine phosphorylation was determined by immunoprecipitation against the ß-subunit of the IR followed by immunoblotting using an anti–phosphothreonine antibody. In both cases, the expression levels of IR were also determined using an anti-insulin receptor ß-subunit antibody.

Next, we asked whether ßAR stimulation, or overexpression of E40K, induces Thr phosphorylation of IR. In WT mice, ISO stimulation induced Thr phosphorylation of IR, and in E40K Tg, Thr phosphorylation was already evident under basal conditions (Figure 7B).

These data suggest that, in vivo, Akt, through Thr phosphorylation of IR, is able to blunt insulin-induced tyrosine phosphorylation of IR.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The findings of this study are that, in neonatal cardiomyocytes, (1) short-term stimulation of ßARs and insulin increase glucose uptake through two different Akt-dependent mechanisms, (2) long-term stimulation of ßARs negatively regulates insulin-induced glucose uptake through inhibition of tyrosine autophosphorylation of IR, and (3) serine phosphorylation of Akt leads to Thr phosphorylation of the IR ß subunit, thereby mediating the ßAR-induced impairment of insulin signaling, and (4) transgenic mice with cardiac overexpression of E40K Akt have impaired insulin-induced tyrosine phosphorylation of IR.

Our results show that ßAR stimulation in cardiomyocytes has a biphasic effect on insulin-stimulated glucose uptake, with an initial additive, followed by an inhibitory action. The additive action on insulin-induced glucose uptake is attributable to the ability of ßAR stimulation to increase glucose uptake using molecular mechanisms distinct from those used by insulin. In fact, our data show that insulin-induced glucose uptake is a PI3K-dependent phenomenon, whereas ISO-induced glucose uptake is a PKA/CaMK/Ca²⁺-dependent and PI3K-independent phenomenon. Our observation is consistent with those previously reported in perfused rat heart,³⁴ in which a Ca²⁺-dependent mechanism was required for both - and ßAR-evoked glucose uptake. In addition, we show that Akt kinase activity is required for ISO-induced glucose uptake in cardiomyocytes. In fact, overexpression of dominant-negative mutant Akt inhibited Akt kinase activity, and this was associated with an inhibition of ISO-induced glucose uptake. Akt is involved in insulin-stimulated glucose uptake,²⁰ and we have previously shown that ISO stimulation activates Akt²⁸ through a CaMK- and PI3K-dependent mechanism. In this study, we report that threonine 308 phosphorylation of Akt peaks at 10 minutes after ISO stimulation, and this is a PKA/CaMK/Ca²⁺-dependent phenomenon. On the other hand, it has been reported³⁵ that phosphorylation in threonine and in serine both stimulate the kinase activity of Akt, and different molecular pathways can account for phosphorylation in threonine or serine. Our finding that ISO-induced threonine 308 phosphorylation of Akt is a PI3K-independent event is consistent with the observation of Alessi et al³⁶ who reported that threonine 308 phosphorylation of Akt is mediated by PDK1, which is insensitive to wortmanin. On the other hand, insulin-evoked glucose uptake as well as threonine 308 phosphorylation of Akt was selectively mediated by a PI3K-dependent pathway. This is in agreement with the observation that insulin-induced glucose uptake can be a calcium-independent phenomenon.³⁷ Thus, the different pathways used by ßAR and insulin in the activation of Akt can account for the additive effect of short-term ISO stimulation on insulin-induced glucose uptake.

Our finding that long-term ßAR stimulation reduces insulin-induced glucose uptake and tyrosine autophosphorylation of IR is partially consistent with that reported by Klein et al²⁴ who showed a reduction of insulin-induced glucose uptake and tyrosine autophosphorylation of IR in cultured adipocytes stimulated with ß₃AR agonist. However, it was reported that this inhibitory effect reached a peak after only 5 minutes, and not after 120 minutes, as we noted in cardiomyocytes, and it is noteworthy that ß₃AR stimulation failed to activate Akt in adipocytes. This reinforces the concept that different ßAR subtypes drive different cell signaling mechanisms, and thus physiology of different cell types depends on expression levels and/or functional coupling with downstream signaling molecules of the ßAR subtypes.

In cultured cardiomyocytes, we found that the reduction of insulin-induced autophosphorylation of IR in response to long-term ßAR stimulation was associated with threonine phosphorylation of the ß-subunit of IR. The presence of several Akt consensus sites in IR and the evidence that, after long-term ßAR stimulation, immunoprecipitates of the ß-subunit of IR are recognized by an anti–phospho-Akt substrate antibody, support the notion that IR is a substrate of Akt. Our data show that long-term ISO stimulation phosphorylates Akt in serine 473, and this is a PKA/CaMK/Ca²⁺- and PI3K-dependent phenomenon. Although our data do not clarify whether or not threonine 308 phosphorylation is required for the subsequent serine 473 phosphorylation of Akt, they allow to speculate that Akt could be the key molecule involved in the ßAR-induced regulation of glucose uptake, mediating both positive and negative feedback on the phosphorylation site (Figure 8).

View larger version (14K): [in this window] [in a new window]   Figure 8. Current hypothesis as to how stimulation of ßAR mediates the cross-talk with the IR in cardiac myocytes. Short-term stimulation of ß-adrenergic receptors through protein kinase A (PKA) and kinase Ca2+-calmodulin–dependent (CaMK) phosphorylates (threonine 308) and activates Akt, which in turn, promotes glucose uptake. Long-term stimulation of ß-adrenergic receptors through a PKA, CaMK, and phosphatidylinositol 3-kinase (PI3K)–dependent pathway phosphorylates (serine 473) and activates Akt, which in turn, phosphorylates the ß subunit of the insulin receptor in threonine. Threonine phosphorylation of the insulin receptor inhibits insulin-induced tyrosine autophosphorylation of ß-subunit of insulin receptor. This mechanism accounts for the impairment of early steps of insulin signaling and, thus, for ß-adrenergic receptor–induced insulin resistance.

The possibility that the PI3K-Akt pathway is involved in the pathogenesis of insulin resistance has been reported by Egawa et al,²¹ who demonstrated that, in adipocytes, overexpression of a constitutively active mutant of PI3K impairs IRS-1 function most likely by serine/threonine phosphorylation. However, Akt is not the only mechanism that accounts for insulin receptor desensitization, because insulin-induced desensitization of its receptor depends also on a tyrosine phosphatases-dependent mechanism (see online data supplement).

In this study, we recognize two potential limitations. In particular, glucose uptake was measured in absence of glucose in the culture media, and the experiments were performed in neonatal rat cardiomyocytes that are not yet fully mature with regards to the control of glucose metabolism and thus have lower expression levels of GLUT4 compared with adult cardiomyocytes.³⁸ However, we found that the signal responsible for ßAR-induced insulin resistance is mediated by ß₁AR subtype (see online data supplement), and cardiomyocytes, in adult rodents as well as in primates, express mainly ß₁AR subtype. Therefore, our observation, is not trivial in terms of biological significance; on the contrary, it is possible that our experimental setting underestimates the phenomenon of ßAR-induced insulin resistance. In addition, we must consider that, consistent with the molecular machinery that account for ßAR stimulation–induced desensitization of IR detected in vitro, adult transgenic mice with cardiac overexpression of constitutively active Akt also showed that Akt is critically involved in the regulation of the first steps of insulin signaling. Therefore, it is possible to speculate that all pathological conditions characterized by sustained stimulation of cardiac ßAR induce a state of insulin resistance in the heart through the activation of Akt.

	Acknowledgments

 
C.M. was supported by a Post-Doctoral fellowship from FEDERICO II University, Naples, Italy. G.C. is a recipient of a Ministero dell’Università e Ricerca Scientifica–Centro Nazionale delle Ricerche (progetto post-genomica; grant no. 499).

	Footnotes

 
Original received December 9, 2003; resubmission received October 25, 2004; revised resubmission received November 17, 2004; accepted November 29, 2004.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

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