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Insulin resistance in adult cardiomyocytes undergoing dedifferentiation: role of GLUT4 expression and translocation ¹

Division of Cardiology, University Hospitals of Geneva, Geneva, Switzerland

Correspondence: Division of Cardiology, University Hospitals of Geneva, 24 Micheli-du Crest, 1211 Geneva 14, Switzerland. E-mail: christophe.montessuit{at}hcuge.ch

SPECIFIC AIMS

To test the hypothesis that cardiac myocytes undergoing dedifferentiation and hypertrophy-like spreading in primary culture develop insulin resistance, we measured basal and insulin-stimulated glucose transport in cardiac myocytes at different time points of phenotypic modification in vitro.

To correlate the insulin-resistant phenotype with expression of glucose transporters, we concomitantly measured expression of the glucose transporters GLUT1 and GLUT4.

To investigate mechanisms leading to insulin resistance, we assessed translocation of glucose transporters to the cardiac myocytes surface in response to insulin and examined activation of signaling pathways known to be involved in control of translocation of GLUT4.

PRINCIPAL FINDINGS

1. Adult rat cardiac myocytes in primary culture develop transient insulin resistance
Adult rat cardiomyocytes (ARC) in primary culture underwent dedifferentiation followed by development of hypertrophy, assessed by morphology and overexpression of atrial natriuretic factor (ANF expression at day 2: 466%±37% of ex vivo; day 10: 5218%±984% of ex vivo). Basal 2-deoxyglucose (2-DG) uptake which refers to glucose uptake in absence of insulin, did not change during the 10 day observation period. Addition of insulin to the medium increased 2-DG uptake 3.2 ± 0.5-fold in ex vivo ARC (P<0.05). However, after 2 and 3 days of culture, insulin-stimulation of 2-DG uptake was completely abolished. On day 7, insulin responsiveness was restored with 3.2 ± 0.5-fold stimulation of 2-DG uptake (P<0.05).

2. Expression of insulin-sensitive glucose transport GLUT4 is reduced
To determine whether insulin-resistance was explained by down-regulation of the insulin-responsive glucose transporter GLUT4, we measured GLUT1 and GLUT4 mRNA and protein levels. The GLUT4/GLUT1 mRNA ratio measured by RT-PCR markedly dropped after 2 days compared with ex vivo ARC (ex vivo: 4.71±0.96 GLUT4 mRNA transcripts/GLUT1 mRNA transcript; day 2: 0.45±0.26 GLUT4 transcripts/GLUT1 transcript) and remained low during the 10 day culture period. Protein content of GLUT4 decreased between the first and second day of culture to 70 ± 7% (P<0.05 vs. ex vivo ARC), remained subsequently constant until day 7 (74±15%) and then decreased further to 37 ± 8% (P<0.05). Protein content of GLUT1 progressively increased during the 10 day culture period to 547 ± 25% (P<0.001). Thus, down-regulation of GLUT4 protein persisted on days 7 and 10 of culture when insulin responsiveness of 2-DG uptake had entirely recovered. Therefore, transient insulin resistance is not readily explained by reduced expression of GLUT4.

3. GLUT4 translocation is impaired in insulin-resistant cardiac myocytes
In 3-days insulin-resistant ARC, no increase of GLUT4 protein was detectable in sarcolemmal sheets in response to insulin, indicating absent translocation of GLUT4 to the sarcolemma (Fig. 1 ). In contrast, after 7 days, when insulin-responsiveness of 2-DG uptake was restored, there was a 3.3-fold increase of GLUT4 protein in sarcolemmal sheets in response to insulin (Fig. 1B ). GLUT1 translocation in response to insulin (1.8-fold) did not change in culture (Fig. 1C ).

View larger version (28K): [in this window] [in a new window]   Figure 1. GLUT4 enrichment in sarcolemmal membrane sheets of insulin-sensitive (7 days) and insulin-resistant (3 days) ARC. A) Representative immunoblots of GLUT4 density in total proteins (TP) and in membrane sheets (MS) of ARC after 3 or 7 days of culture, with and without insulin (18 nM). Insulin resistance in 3 days old ARC is associated with impairment of GLUT4 translocation to the sarcolemma. B–C) Bar diagram of protein ratio membrane sheets (MS)/total protein (TP) for GLUT4 (B) and GLUT1 (C), measured in ARC in absence (white bars) or presence (filled bars) of insulin (18 nM) after 3 or 7 days of culture. Significant increase of the ratio for GLUT4 after 7 days by insulin indicates translocation of GLUT4 to the sarcolemma. Results are mean ± SEM of 4 different experiments. *P < 0.05 vs. the ratio in ARC without insulin stimulation at same time of culture.

4. Phosphorylation of Akt is not reduced in insulin resistance
Activation of the phosphatidylinositol 3-kinase (PI3K)-PDK1-Akt pathway is required for GLUT4 translocation in insulin-responsive cells. In insulin-resistant 3-day-old ARC phosphorylation of Akt in response to insulin was not reduced compared with ex vivo ARC. Accordingly, impairment of activation of the PI3K pathway seems not responsible for insulin resistance.

5. Expression and phosphorylation of Cbl are reduced in insulin-resistant cardiac myocytes
A PI3K -independent pathway contributing to insulin-stimulated GLUT4 translocation involves phosphorylation of proto-oncogene Cbl. Protein expression of Cbl was reduced to 58 ± 13% (P<0.05) in insulin-resistant 3-day-old ARC compared with ex vivo cells (Fig. 2 ). After 7 days of culture, protein expression of Cbl had returned to ex vivo level.

View larger version (29K): [in this window] [in a new window]   Figure 2. Phosphorylated and total Cbl were decreased in insulin-resistant ARC. A) Representative immunoblots showing Cbl phosphorylation in ARC after isolation (ex vivo), or after 3 and 7 days of culture. Phosphorylated proteins were immunoprecipitated using a monoclonal phosphotyrosine antibody. Immunoprecipitates were immunoblotted with Cbl antibody to detect phosphorylated Cbl. The lower part of the blot depicts total protein level of Cbl. B) Protein expression of Cbl in ARC after isolation (ex vivo), and after 3 and 7 days of culture. Photodensity values are mean ± SEM of 5 different experiments and expressed as percentage of the mean value of ex vivo cells. *P < 0.05 vs. ex vivo ARC. C) Effect of contractile arrest by BDM (20 mM) or verapamil (10 µM) on total protein expression of Cbl in 7-day-old ARC without insulin (left panel). Photodensity values are mean ± SEM of 2 different experiments and expressed as percentage of the mean value of control cells. The right panel shows the effect of insulin stimulation on phosphorylation of Cbl in 7-day-old cells with and without contractile arrest. Results are expressed as fold increase of the ratio pCbl/Cbl of cells treated with insulin compared with untreated cells (control). *P < 0.05 vs. control ARC.

Insulin clearly elicited phosphorylation of Cbl in cardiomyocytes ex vivo and after 7 days of culture (Fig. 2) . In contrast, phosphorylation of Cbl was not detectable in 3-day-old ARC.

6. Inhibition of contractile activity reduces both insulin-sensitivity and phosphorylation of Cbl
ARC had contractile activity in vivo and ex vivo, which stopped within 24 h after isolation. Contractile activity resumed spontaneously within 6–7 days in primary culture, presumably because of re-expression of the pacemaker current (I_f). Because duration of insulin resistance coincided with cessation of contractile activity, we determined the effect of inhibiting contraction on insulin-stimulated 2-DG uptake in beating ARC on day 7. Contraction was completely inhibited after addition of either butanedione monoxime (BDM, 20 mM) or verapamil (10 µM).

Contractile arrest induced by either verapamil or BDM treatment did not affect basal 2-DG uptake. However, both BDM and verapamil significantly decreased, but not completely abolished, stimulation of 2-DG uptake by insulin (insulin: 3.6±0.4 fold stimulation; insulin+BDM: 2.2±0.5 fold stimulation; insulin+verapamil: 1.7±0.1 fold stimulation). Neither verapamil nor BDM reduced phosphorylation of Akt elicited by insulin. However, Cbl expression was reduced (–55% by BDM and –66% by verapamil) as well as Cbl phosphorylation (–74% by BDM and –67% by verapamil).

CONCLUSIONS

Results of our study indicate that 1) ARC in culture develop transient insulin resistance which manifests during initial dedifferentiation of cells with disassembly of myofibrils; 2) insulin resistance is not correlated with reduced expression of GLUT4 and recovers spontaneously despite persistent reduction of GLUT4; 3) GLUT4 translocation to sarcolemma is impaired during insulin resistance; 4) among known signaling pathways involved in GLUT4 translocation, activation of phosphatidylinositol 3-kinase by insulin is not compromised, whereas phosphorylation of Cbl is reduced; and 5) contractile arrest may contribute, at least to some extent, to lowering of phosphorylated Cbl.

Insulin resistance in hypertrophied myocardium in vivo has been attributed to a shift from insulin-regulated GLUT4 to less insulin-sensitive GLUT1. This hypothesis is based on observations indicating a reduction of the GLUT4/GLUT1 ratio either at mRNA or protein level. Culturing of ARC elicits partial reversion of differentiated gene expression pattern to a more fetal-like type, similar to overload-hypertrophy and unloading of myocardium in vivo. The present study indicates that glucose transporter isoforms expression follows this general trend. However, dissociation between the time-course of insulin responsiveness and that of reduced expression of GLUT4 suggests a different mechanism for insulin resistance than the shift from GLUT4 to GLUT1.

Recent observations have provided evidence that in adipocytes insulin-receptor-mediated phosphorylation of proto-oncogene protein Cbl, which is independent of the phosphatidylinositol 3-kinase cascade, markedly amplifies translocation of GLUT4 in response to activation of phosphatidylinositol 3-kinase. Our observations suggest that insulin-mediated phosphorylation of Cbl may be defective during transient insulin resistance in ARC. Cessation of beating may contribute to this effect, since pharmacologically induced contractile arrest in beating, insulin-sensitive ARC reduced both 2-DG uptake and phosphorylation of Cbl. ARC in primary culture re-express pacemaker current I_f, which presumably drives contractile activity after day 7 and thereby may contribute to restoration normal Cbl signaling. Another possible, though speculative, explanation for inefficiency of the Akt pathway to induce translocation of GLUT4 is disarray of the actin-based cytoskeletal structure, which occurs in cultured ARC from days 2–5. In adipocytes, GLUT4 translocation is dependent on actin-containing cytoskeletal components. It has recently been shown in adipocytes that translocation of Cbl to the lipid raft activated small GTP binding protein TC10, which elicited stabilization of cortical actin. Whether disarray of actin in ARC and insulin resistance are related to low protein level of Cbl remains to be determined.

View larger version (32K): [in this window] [in a new window]   Figure 3. Regulation of GLUT4 translocation in normal and insulin-resistant cardiac myocytes In fully differentiated cardiac myocytes (A), insulin triggers activation of the PI3K-PDK1-Akt cascade. Insulin also induces phosphorylation of Cbl and its recruitment to a lipid raft-located complex containing flotilin and CAP. Combined activity of both pathways is required for GLUT4 translocation. In dedifferentiated cardiac myocytes (B), activation of the PI3K-PDK1-Akt cascade is normal. However, Cbl is reduced and its phosphorylation is impaired. This prevents translocation of GLUT4, which is also less abundant. In partially redifferentiated, beating cardiac myocytes (C), normal levels of Cbl expression and phosphorylation are restored. Although GLUT4 expression remains low, translocation of a sufficient number of transporters takes place, so that glucose transport increases in response to insulin. An unidentified link seems to exist between contractile activity, whether extrinsically- or intrinsically-driven, and signaling through Cbl.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-1095fje;

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This Article Full Text (PDF) All Versions of this Article: 18/7/872 03-1095fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by ROSENBLATT-VELIN, N. Articles by MONTESSUIT, C. Search for Related Content PubMed PubMed Citation Articles by ROSENBLATT-VELIN, N. Articles by MONTESSUIT, C.