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Endocrinology, doi:10.1210/en.2007-0656
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Endocrinology Vol. 149, No. 3 1064-1074
Copyright © 2008 by The Endocrine Society

Nuclear Receptor Agonists Improve Insulin Responsiveness in Cultured Cardiomyocytes through Enhanced Signaling and Preserved Cytoskeletal Architecture

Christophe Montessuit, Irène Papageorgiou and René Lerch

Division of Cardiology, Geneva University Hospitals, 1211 Geneva 14, Switzerland

Address all correspondence and requests for reprints to: Dr. Christophe Montessuit, Division of Cardiology, Geneva University Hospitals, 24 Micheli-du-Crest, 1211 Geneva 14, Switzerland. E-mail: christophe.montessuit{at}medecine.unige.ch.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Insulin resistance is the failure of insulin to stimulate the transport of glucose into its target cells. A highly regulatable supply of glucose is important for cardiomyocytes to cope with situations of metabolic stress. We recently observed that isolated adult rat cardiomyocytes become insulin resistant in vitro. Insulin resistance is combated at the whole body level with agonists of the nuclear receptor complex peroxisome proliferator-activated receptor {gamma} (PPAR{gamma})/retinoid X receptor (RXR). We investigated the effects of PPAR{gamma}/RXR agonists on the insulin-stimulated glucose transport and on insulin signaling in insulin-resistant adult rat cardiomyocytes. Treatment of cardiomyocytes with ciglitazone, a PPAR{gamma} agonist, or 9-cis retinoic acid (RA), a RXR agonist, increased insulin- and metabolic stress-stimulated glucose transport, whereas agonists of PPAR{alpha} or PPARβ/{delta} had no effect. Stimulation of glucose transport in response to insulin requires the phosphorylation of the signaling intermediate Akt on the residues Thr308 and Ser473 and, downstream of Akt, AS160 on several Thr and Ser residues. Phosphorylation of Akt and AS160 in response to insulin was lower in insulin-resistant cardiomyocytes. However, treatment with 9-cis RA markedly increased phosphorylation of both proteins. Treatment with 9-cis RA also led to better preservation of microtubules in cultured cardiomyocytes. Disruption of microtubules in insulin-responsive cardiomyocytes abolished insulin-stimulated glucose transport and reduced phosphorylation of AS160 but not Akt. Metabolic stress-stimulated glucose transport also involved AS160 phosphorylation in a microtubule-dependent manner. Thus, the stimulation of glucose uptake in response to insulin or metabolic stress is dependent in cardiomyocytes on the presence of intact microtubules.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE CAPACITY of the heart muscle to derive energy from a wide variety of substrates provides the myocardium with remarkable adaptability in the face of the ever-changing metabolic status of the organism. Among the myocardial substrates, glucose accounts for less than 25% of the energy production under normal conditions (1). Glucose is, however, unique among myocardial substrates because 1) energy can be obtained from glucose through glycolysis even in situations of hypoxia or ischemia and 2) ATP obtained from glycolysis is of paramount importance for the maintenance of ionic homeostasis. Indeed, glycolysis-derived ATP seems to be preferentially used for fueling the sarcolemmal ATPases (e.g. Na+/K+-ATPase, Ca2+-ATPase) that maintain the membrane potential and the ion gradients (2, 3). This is particularly important in situations of metabolic stress, as is demonstrated by the poor post-ischemic recovery of function in hearts that do not express the insulin-responsive glucose transporter GLUT4 (4).

The first step of glucose metabolism is transport of glucose across the sarcolemma. In cardiomyocytes, two isoforms of glucose transporter are involved. GLUT1 is located mainly in the sarcolemma under basal conditions and therefore is considered to maintain cellular glucose supply at low insulin concentration (5, 6). GLUT4 on the other hand is mainly located in intracellular membrane compartments and is translocated to the sarcolemma in response to stimuli including increased workload, ischemia, catecholamines, and insulin (7, 8, 9, 10). More recently, activation of the cellular energy gauge AMP-activated protein kinase (AMPK) has also been shown to trigger the translocation of GLUT4 (11).

Insulin resistance is the failure of insulin to stimulate the transport of glucose into its target cells. Myocardial insulin resistance occurs in spontaneously hypertensive rats (SHR) (12) and in patients with coronary artery disease (13) or with left ventricular hypertrophy even in the absence of hypertension (14). Agonists of peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}), mostly thiazolidinediones, are the main treatment currently available that specifically target tissue insulin resistance. PPAR{gamma} are nuclear receptors/transcription factors that function in obligatory heterodimers with the receptor for 9-cis retinoic acid (RA), retinoid X receptor (RXR). The heterodimers are permissive, meaning that they are activated by the binding of either the PPAR{gamma} or the RXR ligand, with binding of both ligands resulting in synergy (15). This suggested that specific RXR ligands, termed rexinoids, could improve insulin sensitivity. Indeed, in vivo studies (16, 17, 18) support this hypothesis. The mechanisms whereby PPAR{gamma} and RXR agonists improve insulin sensitivity at the tissue level remain, however, elusive. In particular, it remains to be demonstrated that PPAR{gamma} and/or RXR activation can have a direct effect on the insulin responsiveness or sensitivity of cardiomyocytes.

We previously observed that cardiomyocytes in primary culture develop insulin resistance that is not directly related to the down-regulation of GLUT4 expression (19). In this study, we investigated the direct effects of PPAR{gamma} and RXR agonists on the insulin responsiveness of cardiomyocytes in primary culture.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
We obtained male Sprague Dawley rats (100–110 g) from IFFA CREDO (L’Arbresle, France). The ethical committee of the Geneva University School of Medicine and the Geneva State Veterinary Office approved the study protocol, which conforms to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Publication No. 85-23, revised 1996).

Adult rat cardiomyocytes (ARC) culture
ARC were isolated by retrograde perfusion of the hearts with collagenase (type II; Biochrom, Oxoid AG, Basel, Switzerland) (20, 21). Dishes were previously coated with 0.1% gelatin for 4 h and incubated overnight with culture medium containing 20% fetal calf serum (FCS). For immunofluorescence studies, cells were plated on laminin-coated glass coverslips. Cells were plated at a density of approximately 20,000 cells/cm2 in M199 medium (Sigma-Aldrich, Buchs, Switzerland) supplemented with 20 mM creatine, 100 µM cytosine-β-D-arabinofuranoside, and 20% FCS. For ex vivo substrate uptake and signaling studies, cells were plated on laminin-coated petri dishes in M199 supplemented with 1% FCS and left to adhere for 2 h before experiments.

The 9-cis RA (Sigma), Wy-14643 (Biomol International, ANAWA, Wangen, Switzerland), L-165041 (Sigma), ciglitazone (Biomol International) and 4-[E-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl]benzoic acid (TTNPB) (Calbiochem, Merck, Volketswil, Switzerland) were added from 1000x ethanol or dimethylsulfoxide stock solutions to the medium at the time of plating (d 0).

Determination of 2-deoxy-D-glucose (2-DG) uptake
Uptake of 2-DG in cardiomyocytes was measured as previously described (22). The cells were incubated for 1 h in the presence or absence of glucose transport agonists, with 2-[6-3H]DG (1–2 µCi/ml), providing a final 2-DG concentration of 10 nM in glucose-free E medium. Preliminary experiments indicated that 2-DG uptake increased linearly with incubation time up to 1 h (supplemental Fig. 1, published as supplemental data on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org). Composition of E medium was 128 mM NaCl, 6 mM KCl, 1.2 mM Na2HPO4/NaH2PO4, 1.4 mM MgSO4, 1 mM CaCl2, 2 mM Na-pyruvate, 10 mM HEPES, and 0.3 mM fatty acid-free BSA, pH 7.4. Uptake was stopped by adding phloretin to a final concentration of 0.4 mM and by immediately washing three times with ice-cold PBS. ARC were then dissolved in 1 ml 0.1 M NaOH, and 10-µl aliquots were taken for protein content determination and the remaining NaOH lysate assayed for radioactivity. Glucose transport agonists used were insulin (10–11 to 10–5 M) and oligomycin (10–6 M). Results are expressed as fold stimulation of 2-DG uptake with respect to control unstimulated cardiomyocytes.

Western blots
After preincubation in serum-free M199 for 30 min, ARC in 6-cm dishes were stimulated by glucose transport agonists for 10 min (insulin) or 20 min (oligomycin). Incubations were stopped by three quick rinses in ice-cold PBS, and ARC were dissolved in 200 µl lysis buffer made of 150 mM NaCl, 50 mM Tris-HCl (pH 7.5), 1 mM Na2EDTA, 0.25% sodium deoxycholate, 1% Igepal CA 630, 5 mM NaF, 10 mM β-glycerophosphate, 10 mM para-nitrophenylphosphate, and 1 mM NaVO3 and supplemented with CompleteMini Protease Inhibitor (Roche Diagnostics AG, Rotkreuz, Switzerland). Proteins (100 µg) were run through 7.5% SDS-PAGE gels and transferred onto polyvinylidene difluoride membranes, which were probed with antibodies for phosphorylated and total signaling intermediates. The following primary antibodies were used: Akt (9272), Akt2 (2962), AMPK{alpha} (2532), insulin receptor (IR)β (3025), phospho-Akt (Ser473) (9271), phospho-(Ser/Thr) Akt substrate (9611), phospho-Akt (Thr308) (9275), phospho-AMPK{alpha} (Thr172) (2532), and phospho-IR (Tyr1150/1151) (3024), from Cell Signaling Technologies (BioConcept, Allschwil, Switzerland); GLUT4 (ab654) from Abcam (Cambridge, UK); and AS160 (07–741) from Upstate Biotechnology (Lucerna Chem, Luzern, Switzerland).

Immunofluorescence analysis of microtubules
ARC cultured on glass coverslips were fixed with 4% paraformaldehyde in PBS and permeabilized with 0.3% Triton X-100 in PBS. After blocking with 10% nonimmune goat serum in PBS, coverslips were incubated overnight at 4 C with a monoclonal anti-{alpha}-tubulin antibody (clone B-5–1-2; Sigma-Aldrich) diluted in PBS containing 1.5% nonimmune goat serum. The coverslips were then incubated with the Alexa Fluor 633-conjugated F(ab')2 fragment of a goat antimouse IgG antibody diluted in PBS containing 1.5% nonimmune goat serum for 1 h at 37 C. Slides were mounted with ProLong Gold (Invitrogen, Basel, Switzerland) and examined with a Carl Zeiss LSM510 confocal microscope. One-micrometer-thick confocal slices running through at least one nucleus were usually acquired. Image luminosity and contrast were digitally enhanced with the ImageJ software (National Institutes of Health, http://rsb.info.nih.gov/ij).

Statistics
Data are presented as mean ± SEM and were compared by ANOVA (Prism 4, GraphPad Software) followed by Bonferroni’s post hoc test. Differences were considered significant when P < 0.05. Dose-response data points were fitted with sigmoid curves by the least-squares method (Prism 4, GraphPad Software).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of nuclear receptor agonists on insulin-stimulated glucose transport
We have previously described that cardiomyocytes develop insulin resistance within 2–3 d in primary culture, whereas insulin responsiveness was restored upon resumption of contractile activity after 6–7 d (19). To determine whether nuclear receptor agonists of the PPAR/RXR heterodimers family directly affected insulin-stimulated glucose transport, cardiomyocytes were incubated for 3 d with pharmacological specific agonists of PPAR{alpha} (Wy-14643), PPARβ/{delta} (L-165041), PPAR{gamma} (ciglitazone), or RXR (9-cis RA). Because 9-cis RA also is an agonist of the retinoic acid receptor (RAR) (15), cardiomyocytes were incubated as well with the RAR-specific ligand TTNPB for comparison. Figure 1Go demonstrates that among the PPAR agonists, only the PPAR{gamma} ligand ciglitazone significantly improved insulin-stimulated glucose uptake in cardiomyocytes. The PPAR{alpha} and PPARβ/{delta} agonists failed to increase glucose uptake (Fig. 1BGo). We verified that 9-cis RA treatment increased insulin-stimulated 2-DG uptake also in the presence of 5.5 mM glucose in M199 (supplemental Fig. 2) or in the presence of various concentrations of cold glucose in E medium (supplemental Fig. 3) (supplemental figures published as supplemental data on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org).


Figure 1
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  		FIG. 1. Some nuclear receptor agonists enhance insulin-stimulated glucose uptake in insulin-resistant cardiac myocytes. A, Basal and insulin-stimulated relative 2-DG uptake in cardiomyocytes cultured for 3 d in the presence of the RXR agonist 9-cis RA (1 µM), the PPAR{gamma} agonist ciglitazone (CG; 10 µM), the RAR agonist TTNPB (10 nM), or the vehicle (C); B, 2-DG uptake in cardiomyocytes cultured for 3 d in the presence of 9-cis RA (1 µM), the PPAR{alpha} agonist Wy-14643 (Wy; 100 µM), the PPARβ agonist L-165041 (L; 10 µM), or the vehicle (C). *, Significant effect of insulin; #, significant effect of the nuclear receptor agonist. Results are mean ± SEM of the number of experiments indicated at the foot of each column.

 
9-cis RA, which is a ligand for all three PPAR/RXR complexes, as well as for the RAR/RXR complex, was the most powerful agent in improving insulin-stimulated glucose transport in cardiomyocytes (Fig. 1Go). Because the effect of 9-cis RA was consistently stronger than that of ciglitazone, we tested whether activation of the RAR could also improve insulin responsiveness. Indeed, treatment of the cardiomyocytes with TTNPB modestly but significantly increased insulin-stimulated glucose uptake.

Next we constructed dose-response curves for the three agents that improved insulin responsiveness. The results indicated that 9-cis RA improved insulin-stimulated glucose transport with an EC50 in the nanomolar range (8.9 x 10–9 ± 2.2 x 10–9 M; mean ± SEM; n = 5) (Fig. 2AGo). Ciglitazone and TTNPB had maximal effects at 10–5 and 10–8 M, respectively (Fig. 2Go, B and C).


Figure 2
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  		FIG. 2. Dose-response curves for nuclear receptor agonists and insulin and additivity of the effects of ciglitazone and TTNPB. A–C, Basal and insulin-stimulated relative 2-DG uptake in cardiomyocytes cultured for 3 d in the presence of various concentrations of 9-cis RA (A), ciglitazone (B), or TTNPB (C); D, basal and insulin-stimulated relative 2-DG uptake in cardiomyocytes cultured for 3 d in the presence of ciglitazone (CG; 10 µM), TTNPB (10 nM), the combination of both, or the vehicle (C); E, 2-DG uptake in cardiomyocytes cultured for 3 d in the presence of ciglitazone (CG; 10 µM), 9-cis RA (1 µM), the combination of both, or the vehicle (C); F, 2-DG uptake in cardiomyocytes cultured for 3 d in the presence of 1 µM 9-cis RA or the vehicle (C) and exposed to various concentrations of insulin. For 9-cis RA, TTNPB, and insulin, dose-response curves were fitted to a sigmoid. *, Significant effect of insulin; #, significant effect of the nuclear receptor agonist(s); §, significantly different from either CG or TTNPB alone. Numbers at the foot of each column indicate the number of experiments.

 
9-cis RA is an agonist for both the PPAR{gamma}/RXR and the RAR/RXR complexes, which are separately activated by ciglitazone and TTNPB, respectively. Thus, we reasoned that if the larger improvement of insulin responsiveness by 9-cis RA is due to its acting on both complexes simultaneously, then the effect of maximal doses of ciglitazone and TTNPB should be additive. As shown in Fig. 2DGo, the stimulation of glucose transport by insulin in cardiomyocytes treated with a combination of 10–5 M ciglitazone and 10–8 M TTNPB was larger than in cardiomyocytes treated with either agent alone. In contrast, the effects of 10–5 M ciglitazone and 10–6 M 9-cis RA on insulin responsiveness were not additive (Fig. 2EGo).

The insulin-stimulated glucose transport process is characterized in terms of insulin sensitivity and insulin responsiveness. Insulin sensitivity is defined in relation to EC50, the concentration of insulin required to induce 50% of its maximal effect on glucose transport, whereas insulin responsiveness is defined as the increase in glucose transport induced by a maximally effective insulin concentration. Experiments described above were performed with a supraphysiological dose of insulin; hence, the results suggested that nuclear receptor agonist treatment augmented insulin responsiveness. To determine whether treatment of cardiomyocytes with 9-cis RA also altered insulin sensitivity, we constructed dose-response curves for insulin in cardiomyocytes after 3 d in culture (Fig. 2FGo). The results confirmed that 9-cis RA treatment improved insulin responsiveness; insulin sensitivity was not significantly affected by treatment with 9-cis RA (EC50 control, 3.5 ± 0.6 nM vs. 9-cis RA, 2.9 ± 0.9 nM; n = 5). It should be noted, however, that insulin sensitivity was reduced in cultured cardiomyocytes compared with ex vivo, freshly isolated cells (EC50 ex vivo, 0.19 ± 0.06 nM; n = 5).

Insulin signaling in insulin-responsive and insulin-resistant cardiomyocytes
The absence of effect of the 9-cis RA treatment on insulin sensitivity suggested that nuclear receptor agonists did not influence expression of the IR or its activation in response to insulin. Indeed, we observed in Western blot experiments that the expression of IR was constant during culture of cardiomyocytes and was not affected by nuclear receptor agonist treatment (Fig. 3Go). The phosphorylation of IR on key tyrosine residues in response to insulin was reduced in 3-d insulin-resistant cardiomyocytes but restored in 9-cis RA-treated cardiomyocytes.


Figure 3
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  		FIG. 3. Effect of nuclear receptor agonists on insulin signaling in cultured cardiomyocytes. A, Expression and phosphorylation state of IRβ, Akt, and AS160 in response to insulin in ex vivo or cultured cardiomyocytes. Note that the Akt2 antibody used less efficiently recognized activated Akt2. B–E, Semiquantitative determination of the phosphorylation of IRβ on residues Tyr 1150/1151 (B), Akt on residue Ser473 (C), Akt on residue Thr308 (D), and AS160 on Akt target residues, i.e. Ser 318/341/570/588/751 and Thr642 (E). Results are expressed relative to d-3 control stimulated with insulin, arbitrarily set at 1. *, Significant effect of insulin; #, significantly different from d-3 control.

 
Akt is a key downstream effector of insulin signaling in glucose transport regulation, which becomes fully activated by sequential phosphorylation on Ser473 and Thr308 residues (23). We have previously observed that phosphorylation of Ser473 was normal in insulin-resistant cardiomyocytes in primary culture (19). In the present study, 3-d cardiomyocytes with reduced insulin responsiveness exhibited very low phosphorylation of Thr308 upon stimulation with insulin, compared with ex vivo or 7-d insulin-responsive cells (Fig. 3Go). Phosphorylation of Akt on Ser473 was also significantly reduced, although proportionally less than Thr308. Treatment of the cardiomyocytes with 9-cis RA markedly improved insulin-induced Akt phosphorylation on both sites. Ciglitazone and TTNPB improved Akt phosphorylation to a lesser extent than 9-cis RA. Akt exists in three isoforms expressed in the heart, of which Akt2 is responsible for mediating the metabolic effects of insulin (24, 25). We verified that chronic treatment with 9-cis RA did not affect Akt2 expression in cardiomyocytes. Western blots with either a pan-Akt antibody or an Akt2-specific antibody showed no differences in Akt expression over time or in the presence of 9-cis RA.

The Rab GTPase-activating protein AS160 has recently been identified a substrate of Akt (26, 27), which upon phosphorylation dissociates from GLUT4-containing vesicles, a required step for the insertion of GLUT4 in the plasma membrane (28). Accordingly, we observed phosphorylation of AS160 in response to insulin in ex vivo cardiomyocytes (Fig. 3Go). Assessment of AS160 expression during culture of cardiomyocytes proved to be difficult to interpret, because we observed that the AS160 antibody 07-741, which is supposed to detect AS160 independently of its phosphorylation state, depending on batches, sometimes better recognized phosphorylated AS160 (supplemental Fig. 4, published as supplemental data on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org). Therefore we decided to normalize quantitation of AS160 phosphorylation by the expression of its upstream kinase Akt, the expression of which did not change with culture conditions. By d 3, insulin-stimulated phosphorylation of AS160 was reduced in untreated cardiomyocytes. Chronic treatment with 9-cis RA resulted in increased phosphorylation of AS160 in response to insulin. A less pronounced improvement in AS160 phosphorylation could be observed in cardiomyocytes exposed to ciglitazone in culture, whereas TTNPB induced a larger but highly variable improvement of AS160 phosphorylation in response to insulin.

Role of microtubules in insulin responsiveness in cardiomyocytes
Translocation of GLUT4 vesicles to the cell surface in 3T3-L1 adipocytes is highly dependent on the microtubule network (29, 30), which is built up upon stimulation with insulin (31). In contrast, disruption of the microtubule network in skeletal muscle myocytes does not affect insulin-stimulated glucose transport (32). Because we previously observed a beneficial effect of 9-cis RA on the morphology of the cytoskeleton in cultured cardiomyocytes (33), we investigated the relationship between insulin responsiveness and microtubule morphology in cardiomyocytes. In insulin-responsive cardiomyocytes, either ex vivo or cultured for 3 d with 9-cis RA, disruption of microtubules with colchicine completely abolished insulin responsiveness in terms of glucose transport (Fig. 4Go, A and B). Insulin signaling was intact at the levels of the IR and Akt, but phosphorylation of AS160 was reduced in cardiomyocytes with disrupted microtubules (Fig. 4Go, C–H), more prominently so in cardiomyocytes cultured with 9-cis RA. Ex vivo cardiomyocytes exhibited a well-developed microtubule network already before insulin stimulation, with characteristic perinuclear microtubule-organizing centers (MTOC) (34, 35) (Fig. 5AGo). We could not detect differences in the morphology of the microtubule network after insulin stimulation by our static imaging approach (data not shown). However, although the morphology of the microtubule network looked normal after paclitaxel treatment of ex vivo cardiomyocytes, insulin-stimulated glucose uptake was completely abolished. This suggests that some remodeling of the microtubule network occurs in response to insulin and is required for stimulation of glucose uptake.


Figure 4
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  		FIG. 4. Importance of the microtubule network for insulin-stimulated glucose uptake in cardiomyocytes. A and B, Basal and insulin-stimulated 2-DG uptake in ex vivo cardiomyocytes (A) or cardiomyocytes cultured for 3 d in the presence of 9-cis RA (B) in which the microtubule network was either disrupted with colchicine (1 µM; 1 h) or stabilized with paclitaxel (10 µM; 1 h); C and D, insulin signaling in ex vivo cardiomyocytes (C) or cardiomyocytes cultured for 3 d in the presence of 9-cis RA (D) in which the microtubule network was either disrupted with colchicine (1 µM; 1 h) or stabilized with paclitaxel (10 µM; 1 h); E–H, semiquantitative determination of the phosphorylation of IRβ on residues Tyr 1150/1151 (E), Akt on residue Ser473 (F), Akt on residue Thr308 (G), and AS160 on Akt target residues, i.e. Ser 318/341/570/588/751 and Thr642 (H). Results are expressed relative to control stimulated with insulin, arbitrarily set at 1. *, Significant effect of insulin; §, significant effect of the microtubule-perturbing drug.

 

Figure 5
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  		FIG. 5. Morphology of the microtubules in cardiomyocytes. A, Confocal images of the microtubule network in ex vivo cardiomyocytes exposed to either colchicine or paclitaxel, fixed, and stained with an anti-{alpha}-tubulin antibody. Bar, 10 µM. B, Confocal images of the microtubule network in cardiomyocytes cultured for 3 d in the presence or absence of 9-cis RA. Bar, 10 µm.

 
Control 3-d cardiomyocytes showed a disrupted microtubule network. In particular, the MTOC were often fainter or altogether absent compared with ex vivo cardiomyocytes (Fig. 5BGo). In contrast, 9-cis-RA-treated cardiomyocytes displayed a better preservation of the microtubule network, including the MTOC.

Effect of nuclear receptor agonists on AMPK-stimulated glucose uptake and AMPK signaling
Stimulation of glucose transport by translocation of GLUT4 also occurs in response to metabolic stress. The AMPK cascade controls this insulin-independent effect (11, 36). We observed that stimulation of glucose uptake by oligomycin, which strongly activates the AMPK cascade but not insulin signaling, was also markedly enhanced in 9-cis-RA-treated cardiomyocytes (Fig. 6AGo) and to a lesser extent in ciglitazone- or TTNPB-treated cardiomyocytes. Activation of the AMPK cascade converges with insulin signaling onto AS160, which is a substrate for AMPK-mediated phosphorylation (37, 38). In fact, we observed an improved phosphorylation of AS160 in response to oligomycin in 9-cis-RA-treated cardiomyocytes (Fig. 6Go, B–D). Phosphorylation of AS160 in cardiomyocytes treated with ciglitazone was similar to that in untreated cardiomyocytes, despite the inhibitory effect of chronic ciglitazone exposure on AMPK phosphorylation. Similar to insulin-stimulated glucose transport, oligomycin-stimulated glucose transport was dependent on the presence of an intact and plastic microtubular network (Fig. 6EGo), and phosphorylation of AS160, but not AMPK, in response to oligomycin was reduced when microtubules were disrupted with colchicine or stabilized with paclitaxel (Fig. 6FGo).


Figure 6
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  		FIG. 6. Effect of nuclear receptor agonists on AMPK-stimulated glucose transport in cardiomyocytes. A, Basal and oligomycin-stimulated relative 2-DG uptake in cardiomyocytes cultured for 3 d in the presence of the RXR agonists 9-cis RA (1 µM), the PPAR{gamma} agonist ciglitazone (CG; 10 µM), the RAR agonist TTNPB (10 nM), or the vehicle (C). B, AMPK signaling in cardiomyocytes cultured for 3 d in the presence of 1 µM 9-cis RA, 10 µM CG, 10 nM TTNPB, or the vehicle (C). p(S/T) AS160, Phosphorylated AS160 detected with the phospho-(Ser/Thr) Akt substrate antibody. C and D, Semiquantitative determination of the phosphorylation of AMPK on residue Thr 172 (C) and AS160 on AMPK target residues, i.e. Ser 318/341/570/588/751 (D). E, 2-DG uptake in cardiomyocytes cultured for 3 d in the presence of 9-cis RA in which the microtubule network was either disrupted with colchicine (1 µM; 1 h) or stabilized with paclitaxel (10 µM; 1 h). F, AMPK signaling in cardiomyocytes cultured for 3 d in the presence on 9-cis RA in which the microtubule network was either disrupted with colchicine (1 µM; 1 h) or stabilized with paclitaxel (10 µM; 1 h). *, Significant effect of oligomycin; #, significant effect of the nuclear receptor agonist; §, significant effect of the microtubule-perturbing drug.

 
Expression of glucose transporters
Stimulation of glucose transport by insulin occurs through recruitment of the glucose transporters GLUT4 and to a minor extent GLUT1. Previous studies form our laboratory had shown that GLUT4 expression was reduced and GLUT1 expression increased in cultured cardiomyocytes (19, 39). Although our previous experiments suggested that the development of insulin resistance in vitro was independent of GLUT4 expression (19), we determined whether changes in GLUT4 expression in response to nuclear receptor agonists could contribute to the improved insulin responsiveness. Similar to published observations (33, 39), GLUT4 expression was reduced after 3 d in culture but slightly enhanced in the presence of 9-cis RA or TTNPB (Fig. 7Go). Ciglitazone had no significant effects on expression of GLUT4.


Figure 7
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  		FIG. 7. Expression of the glucose transporter GLUT4 in cultured cardiomyocytes. A, Expression of the glucose transporter protein GLUT4 in freshly isolated cardiomyocytes (ex vivo) or cardiomyocytes cultured for 3 d in the presence of 9-cis RA (1 µM), ciglitazone (CG; 10 µM), TTNPB (10 nM), or the vehicle (C); B, semiquantitative determination of GLUT4 expression. *, Significantly different from d-3 control.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The main finding of this study is that nuclear receptor agonists can have a direct positive effect on the insulin responsiveness of cardiomyocytes. Enhanced insulin signaling is responsible for the amelioration of insulin responsiveness and is at least in part due to preservation of the cytoskeletal architecture.

Nuclear receptors involved
The identity of the nuclear receptor(s) mediating the effects of 9-cis RA on glucose uptake is not readily apparent from the data obtained in this study. Being the physiological ligand of the RXR, 9-cis RA has the potential to activate several nuclear receptor/transcription factor heterodimers with permissive activation properties (15). Nuclear receptors forming permissive heterodimers with RXR include PPAR{alpha}, PPARβ/{delta}, and PPAR{gamma}. Our observations strongly suggest the involvement of PPAR{gamma} but rule out the other two PPAR isoforms. In addition, a role for the RAR is suggested by the improvement of insulin-stimulated glucose uptake in cardiomyocytes treated with TTNPB, a specific RAR agonist (40). Although the RAR/RXR heterodimer is not permissive, i.e. binding of 9-cis RA to RXR does not activate it, 9-cis RA is also an activating ligand for RAR (41). The additivity of the effects of ciglitazone and TTNPB, selective agonists of PPAR{gamma} and RAR, respectively, which do not dimerize with one another, confirms that activation of at least the RXR/PPAR{gamma} and RXR/RAR complexes can improve insulin responsiveness in cardiac myocytes. This does not rule out other 9-cis RA-responsive nuclear receptor complexes such as RXR/RXR homodimer, and other RXR/nuclear receptor heterodimers, because the improvement in insulin responsiveness brought about by 9-cis RA alone is more pronounced and robust as that induced by the combination of ciglitazone and TTNPB.

We observed a biphasic dose-response curve for ciglitazone, which would indicate two separate mechanisms of action. High to very high doses (10–5 to 3 x 10–4 M) of two other thiazolidinediones, rosiglitazone (42) and troglitazone (43), have been shown to activate AMPK activity in muscle cells. In turn, chronic AMPK activation has recently been demonstrated to enhance insulin responsiveness in C2C12 myotubes, in relation with increased basal phosphorylation of Akt on Thr308 (44). We could indeed detect changes in phospho-Thr308 Akt in ciglitazone-treated cardiomyocytes stimulated with insulin; basal Thr308 phosphorylation was, however, unchanged. Furthermore, we observed that chronic ciglitazone treatment of cardiomyocytes resulted in blunted AMPK activation. Thus, it seems unlikely that ciglitazone acted through chronic AMPK activation. Alternatively, chronic treatment of diabetic mice with rosiglitazone ameliorated ex vivo insulin-stimulated glucose uptake in skeletal muscle without improvement in Akt phosphorylation (18). In this case, increased insulin responsiveness was attributed to increased signaling via the CAP (c-Cbl-associated protein)/c-Cbl pathway, which has been proposed to be required for insulin responsiveness independently of Akt activation (45). In a previous study, we had observed a defective CAP/c-Cbl signaling in cardiomyocytes that were insulin resistant after 3 d in culture (19). In the present study, we could not observe restoration of CAP/c-Cbl signaling by treatment with ciglitazone. More recent studies have cast serious doubts on the importance of the CAP/c-Cbl pathway for insulin action on glucose transport (46, 47).

Role of the microtubules network
The importance of an intact and dynamic microtubule network for the stimulation of glucose transport by insulin has been extensively studied in adipocytes (29, 30, 31). In contrast, the microtubule network does not appear to be important in skeletal muscle myocytes (32). Cardiomyocytes stand somehow in between these two cell types; similar to skeletal myocytes, an extensive microtubules network preexists to insulin stimulation, but similar to adipocytes, disruption of the network completely prevents insulin-mediated stimulation of glucose uptake. In adipocytes, disruption of the microtubule network results in partial disorganization of insulin signaling. Whereas phosphorylation of Akt on Ser473 is normal in adipocytes in which the microtubules network has been disrupted by nocodazole treatment, phosphorylation on Thr308 is markedly reduced (48). This situation matches our observations in cultured cardiomyocytes, which exhibited a more severe deficit in Akt phosphorylation on Thr308 than on Ser473 associated with an abnormal morphology of the microtubules network, both defects being corrected by treatment with 9-cis RA. Our observations, however, diverge in that Thr308 phosphorylation in response to insulin was unaltered either in ex vivo or 9-cis RA-treated cardiomyocytes in which microtubules had been destroyed with colchicine.

Interestingly, disruption of the microtubular network results in reduced phosphorylation of the Rab GTPase-activating protein AS160. The exact role of AS160 in the regulation of GLUT4 trafficking remains highly controversial. According to current models of insulin-stimulated GLUT4 translocation, AS160 is associated with the intracellular GLUT4-containing vesicles, where it keeps small G proteins of the Rab family in an inactive state by promoting GTP hydrolysis (27). This inhibition is relieved when AS160 is phosphorylated by Akt and/or AMPK. Rab proteins regulate several steps of membrane transport, including vesicle budding, motility, tethering, and fusion. Whether AS160 regulation of Rab is important for GLUT4 vesicle fusion, upstream for GLUT4 vesicle movement from the cell core to the periphery or both remains unknown. Experiments performed with cell-free adipocyte extracts indicate that insulin-stimulated Akt activity is required for fusion of GLUT4 vesicles with the plasma membrane, regardless of how GLUT4 vesicles came to the vicinity of the plasma membrane (49). It is tempting to speculate that disruption of the microtubules prevents GLUT4-containing vesicles, and therefore AS160, from coming into contact with the insulin-signaling complex near the cell surface. In fact, several studies have observed GLUT4-containing vesicles moving along microtubules in adipose cells, even in the absence of insulin stimulation, either to and from the cell core and the periphery (50) or beneath the plasma membrane (51). However, because of the aforementioned limitations in determining AS160 expression, we cannot entirely rule out increased AS160 expression as a component of the improved coupling between Akt and AS160.

The mechanism(s) by which treatment with 9-cis RA maintains the integrity of the microtubules remains unknown. Differentiation of P19 embryonal carcinoma cells along the neural pathway induced by RA is associated with stabilization of the microtubules. This appears related both to the expression of specific microtubule-associated proteins and to concomitant posttranslational modifications of tubulin (52). Whether 9-cis RA influences expression of microtubule-associated proteins in cardiomyocytes remains to be investigated. It is important to note that although 9-cis RA preserves the microtubular network, it probably does not do so at the expense of its plasticity. This conclusion is drawn from the opposite effects of paclitaxel, which stabilizes microtubules but also prevents their remodeling, and 9-cis RA on insulin-stimulated glucose uptake.

Role of glucose transporters expression
In a previous study, we observed that 9-cis RA stimulated GLUT4 protein expression in cardiomyocytes with a biphasic dose-response curve and a much smaller EC50 (33). Thus, the improvement of insulin responsiveness exerted by 9-cis RA in this study is unlikely to result mostly from increased GLUT4 expression. In fact, GLUT4 protein expression was only slightly increased in response to 1 µM 9-cis RA, the magnitude of this effect being insufficient to explain restored insulin responsiveness. Similarly, ciglitazone did not improve insulin responsiveness by increasing the expression of GLUT4. This thiazolidinedione was shown to be without effect on GLUT4 expression in this study as in H9c2 cardiomyoblasts (53) and neonatal rat cardiomyocytes (54). Finally, in our previous study, TTNPB was found to be a very potent agonist of GLUT4 protein expression in low FCS culture conditions (33). In this series of experiments performed in high FCS culture conditions, we could detect only a modest increase in GLUT4 protein expression in TTNPB-treated cardiac myocytes.

The glucose transporter GLUT1 is also expressed in cardiomyocytes, and its expression in increased in the culture conditions used in this study (39). In contrast to GLUT4, the majority of GLUT1 resides at the cell surface in unstimulated cells; nevertheless, a fraction of GLUT1 also exists in intracellular vesicles together with GLUT4 and is recruited to the cardiomyocyte surface upon insulin stimulation (55). It is therefore possible that the improved insulin responsiveness observed in nuclear receptor-treated cardiomyocytes results from increased GLUT1 expression in the context of unameliorated GLUT1/4 translocation. However, because the majority of GLUT1 resides at the cell surface in unstimulated cardiomyocytes (56), a substantial overexpression of GLUT1 would proportionally manifest itself more as an increased basal than insulin-stimulated glucose uptake. Of note, basal glucose uptake did tend to increase in 9-cis RA-treated cardiomyocytes, but proportionally much less than insulin-stimulated glucose uptake.

Study limitations
The mechanism(s) by which nuclear receptor agonists improve insulin signaling and preserve microtubule integrity remains unknown. Most biological effects of nuclear receptors stem from their transcription factor functions. However, it is not clear the expression of what protein(s) is regulated to improve insulin responsiveness in this model. Expression of all the signaling intermediates investigated was not changed in response to nuclear receptor agonists. Therefore, the target protein responsible for increased insulin responsiveness in this model is not known. Possible candidates include microtubule-associated proteins that participate in the stabilization of the microtubules.

It is worth noting that, among nuclear receptors, PPAR{gamma} and RAR can inhibit nuclear factor-{kappa}B activity by forming inactive complexes with p65/p50 (57, 58). In L6 myotubes, fatty acid-induced nuclear translocation of nuclear factor-{kappa}B was shown to decrease insulin-stimulated glucose transport independently of Akt phosphorylation (59). Whether such an effect takes place in cultured cardiomyocytes in response to nuclear receptor agonists remains to be investigated.

In a previous publication, we attributed the development of insulin resistance to an impairment of Cbl signaling associated with cessation of contractile activity (19). The present study indicates that the most likely culprit is disruption of the microtubular network. Whether or not this mechanism is relevant for insulin resistance that has been observed in vivo, including in cardiac patients, remains to be investigated. Heart hypertrophy is associated with increased stability of the microtubules, which, according to our data obtained with paclitaxel, may result in insulin resistance if the increased stability is at the expense of the plasticity. Interestingly, a recent abstract suggests that hypertrophy is also associated with microtubular disarray, impeding the movement of membrane vesicles within the cardiomyocytes (60).


   Acknowledgments
 
We thank Laurent Derouette for conducting pilot experiments and Christelle Viglino for excellent technical help.


   Footnotes
 
This study was supported by grants from the Swiss Cardiology Foundation (To C.M.), the Fondation Carlos et Elsie de Reuter (no. 414 to C.M.), the Swiss National Science Foundation (3200B-108238 to C.M. and 3100B0-109212 to R.L.), and the Swiss Cardiovascular Research and Training Network (to C.M. and R.L.).

Disclosure Statement: The authors have nothing to disclose.

First Published Online December 6, 2007

Abbreviations: AMPK, AMP-activated protein kinase; ARC, adult rat cardiomyocytes; CAP, c-Cbl-associated protein; 2-DG, 2-deoxy-D-glucose; FCS, fetal calf serum; IR, insulin receptor; MTOC, microtubule-organizing center; PPAR{gamma}, peroxisome proliferator-activated receptor {gamma}; RA, retinoic acid; RAR, retinoic acid receptor; RXR, retinoid X receptor; TTNPB, 4-[E-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl]benzoic acid.

Received May 17, 2007.

Accepted for publication November 26, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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