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Endocrinology Vol. 139, No. 11 4531-4539
Copyright © 1998 by The Endocrine Society

ARTICLES

The New Antidiabetic Drug MCC-555 Acutely Sensitizes Insulin Signaling in Isolated Cardiomyocytes1

Li Sen Liu, Hideho Tanaka, Shinichi Ishii and Jürgen Eckel

Molecular Cardiology, Diabetes Research Institute, Düsseldorf, Germany; and Mitsubishi Chemical Co. (H.T., S.I.), Yokohama, Japan

Address all correspondence and requests for reprints to: Prof. Dr. Jürgen Eckel, Diabetes Research Institute, Auf’m Hennekamp 65, D-40225 Düsseldorf, Germany. E-mail: eckel{at}uni-duesseldorf.de


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Freshly isolated adult rat ventricular cardiomyocytes have been used to characterize the action profile of the new thiazolidinedione antidiabetic drug MCC-555. Preincubation of cells with the compound (100 µM for 30 min or 10 µM for 2 h) did not modify basal 3-O-methylglucose transport, but produced a marked sensitizing effect (2- to 3-fold increase in insulin action at 3 x 10-11 M insulin) and a further enhancement of maximum insulin action (1.8-fold). MCC-555 did not modulate autophosphorylation of the insulin receptor and tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1). However, insulin action (10-10 and 10-7 M) on IRS-1-associated phosphatidylinositol (PI) 3-kinase activity was enhanced 2-fold in the presence of MCC-555. Association of the p85 adapter subunit of PI 3-kinase to IRS-1 was not modified by the drug. Immunoblotting experiments demonstrated expression of the peroxisomal proliferator-activated receptor-{gamma} in cardiomyocytes reaching about 30% of the abundance observed in adipocytes. The insulin-sensitizing effect of MCC-555 was lost after inhibition of protein synthesis by preincubation of the cells with cycloheximide (1 mM; 30 min). Cardiomyocytes from obese Zucker rats exhibited a completely blunted response of glucose transport at 3 x 10-11 M insulin. MCC-555 ameliorates this insulin resistance, producing a 2-fold stimulation of glucose transport, with maximum insulin action being 1.6-fold higher than that in control cells. This drug effect was paralleled by a significant dephosphorylation of IRS-1 on Ser/Thr. In conclusion, MCC-555 rapidly sensitizes insulin-stimulated cardiac glucose uptake by enhancing insulin signaling resulting from increased intrinsic activity of PI 3-kinase. Acute activation of protein expression leading to a modulation of the Ser/Thr phosphorylation state of signaling proteins such as IRS-1 may be underlying this process. It is suggested that MCC-555 may provide a causal therapy of insulin resistance by targeted action on the defective site in the insulin signaling cascade.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE THIAZOLIDINEDIONES (TZD) represent a new family of orally active antidiabetic drugs that increase the insulin sensitivity of all target tissues (for review, see Refs. 1, 2). A variety of studies in genetic (3, 4) and nongenetic (5) rodent models of insulin resistance have confirmed that these compounds reduce plasma glucose, insulin, and triglyceride levels. However, TZD are ineffective in euglycemic animals; thus, the induction of hypoglycemia does not occur (2). In vivo, insulin sensitizing was shown to increase glucose disposal and reduce hepatic glucose output in rodent models of insulin resistance (6, 7). In vitro, TZD increase glucose uptake and glucose transporter expression in adipocytes (8) and myocytes (9) and inhibit gluconeogenesis in hepatocytes (10). Clinical trials have confirmed the efficacy of TZD for the treatment of NIDDM patients (for review, see Ref. 2). Further, the drugs may also be suitable for treatment of nondiabetic insulin-resistant states such as obesity (11), the polycystic ovarian syndrome (12), and Werner’s syndrome (13).

Despite their high impact on the future treatment of insulin-resistant patients, the molecular pathways of TZD action have remained poorly understood. At least three different models may be considered. 1) Convincing evidence supports the idea that the transcription factor peroxisomal proliferator-activated receptor-{gamma} (PPAR{gamma}) represents the intracellular receptor for TZD action (14), with the in vivo efficacy of TZD as antidiabetic drugs being correlated to their potency as PPAR{gamma} agonists in vitro (15). In this model, the insulin sensitization would involve transcriptional regulation and enhanced expression of genes representing critical components of the insulin signaling cascade (1, 2). Consistently, most TZD require long term treatment, and a number of fat cell-specific genes and additionally GLUT4 (8) have been shown to be controlled by these drugs (16) in agreement with high expression of PPAR{gamma} in this tissue (17). However, TZD-responsive genes of the insulin signaling cascade have not been identified to date. 2) We (9) and others (18) have shown that TZD may be able to modulate protein kinase C (PKC) activity in muscle tissue. This involves acute inhibition of membrane-associated PKC isoforms (9) as well as reversal of chronic alterations of PKC in fat-fed rats (18, 19). As serine phosphorylation of early signaling components may play a pivotal role in the pathogenesis of insulin resistance (20), modulation of PKC by TZD may represent an additional, yet less defined, pathway for the action of these compounds. 3) More recent studies by Berger and co-workers (21, 22) suggest that pioglitazone and several other TZD may be able to directly potentiate insulin signaling at the level of phosphatidylinositol (PI) 3-kinase in Chinese hamster ovary cells overexpressing human insulin receptors and in adipose tissue. However, these findings were not confirmed in 3T3-L1 adipocytes (23). Further, the precise mechanism underlying PI 3-kinase modulation by TZD remains obscure.

Using isolated rat cardiomyocytes, we have now characterized the action profile of a novel TZD drug, MCC-555 (Fig. 1Go). In insulin-resistant rodent models, this compound was shown to be 5 times more potent than pioglitazone (24). We therefore investigated whether MCC-555 was able to modify insulin signaling in a primary muscle cell and if this might serve to ameliorate insulin resistance in myocytes from obese Zucker rats (20). The data show that MCC-555 acutely sensitizes insulin action by increasing the intrinsic activity of insulin receptor substrate-1 (IRS-1)-associated PI 3-kinase activity. The drug effect was dependent on protein synthesis and may involve the PPAR{gamma} pathway.



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  		Figure 1. Structure of MCC-555.

 

   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals
3-O-[Methyl-14C]D-glucose ([methyl-14C]3-OMG; SA, 57 Ci/mmol), L-[1-14C]glucose (SA, 58 Ci/mmol), [33P]orthophosphate (SA, 3000 Ci/mmol), and 125I-labeled protein A (30 mCi/mg) were obtained from Amersham (Braunschweig, Germany). [{gamma}-32P]ATP (SA, 6000 Ci/mmol) was purchased from New England Nuclear (Dreieich, Germany). Collagenase was a product of Serva (Heidelberg, Germany). Wortmannin, cycloheximide, and PI were supplied by Sigma Chemical Co. (Munich, Germany). Polyclonal anti-p85 antibodies as well as the monoclonal antiphosphotyrosine antibody coupled to alkaline phosphatase were obtained from Upstate Biotechnology, Inc. (Lake Placid, NY). The anti-IRS-1 antibody was provided by Dr. Ton Maassen (Leiden, The Netherlands). The antiinsulin receptor antibody was obtained from Transduction Laboratories (Lexington, MA). Polyclonal PPAR{gamma} antiserum was provided by J. Holder (Epsom, UK). Protein A- and protein G beads were supplied by Pierce Chemical Co. (Cologne, Germany). MCC-555 was provided by Mitsubishi Chemical Co. (Yokohama, Japan). All other chemicals were of the highest analytical grade and were purchased from Sigma Chemical Co. or Merck (Darmstadt, Germany).

Isolation of ventricular cardiomyocytes
Ca2+-tolerant myocytes were isolated from male Wistar rats (260–320 g) or genetically obese (fa/fa) male Zucker rats (480–520 g) by perfusion of the heart with collagenase as previously described by us (25, 26). All animal experimentation was performed according to approved protocols. The final cell suspension was washed three times with HEPES buffer (130 mM NaCl, 4.8 mM KCl, 1.2 mM KH2PO4, 25 mM HEPES, 5 mM glucose, and 20 g/liter BSA, pH 7.4, equilibrated with oxygen) and incubated in silicone-treated Erlenmeyer flasks in a rotating water bath shaker at 37 C. After 20 min, CaCl2 and MgSO4 (final concentration, 1 mM) were added, and incubation was continued for at least 60 min. Cell viability was checked by determination of the percentage of rod-shaped cells and averaged 90–95% under all incubation conditions. For labeling with [33P]orthophosphate, freshly prepared cardiomyocytes were incubated in phosphate-free HEPES buffer with 100 µCi [33P]orthophosphate/ml cell suspension (1 x 106 cells/ml) for 2.5 h, as recently described (20).

Assay of 3-OMG transport
Transport experiments were performed at 37 C in HEPES buffer containing MgCl2 (1 mM) and CaCl2 (1 mM). The reaction was started by pipetting a 50-µl aliquot of the cell suspension to 50 µl HEPES buffer containing [methyl-14C]3-OMG (final concentration, 100 µM). Carrier-mediated glucose transport was then determined using a 10-sec assay period and L-[14C]glucose to correct for simple diffusion as described in earlier reports from this laboratory (9, 20).

Immunoprecipitation
Cells were treated with different doses of MCC-555, stimulated with insulin as indicated, and lysed in a buffer containing 50 mM Tris, 150 mM NaCl, 20 mM NaF, 10 mM EDTA, 1 mM Na3VO4, 0.3 mM phenylmethylsulfonylfluoride, 2 µM leupeptin, 2 µM pepstatin, 4 trypsin inhibitor units/ml aprotinin, and 1% (vol/vol) Triton, pH 7.4. After incubation for 1 h, the suspension was centrifuged at 16,000 x g for 5 min. After this, the supernatant was subjected to immunoprecipitation. For immunoprecipitation of IRS-1, the antiserum was preadsorbed to protein A/protein G beads for 2 h at 4 C, then added to the solubilized cell supernatant and incubated for 16 h at 4 C with gentle rotation. After centrifugation, the immunopellet was washed three times with lysis buffer and twice with PBS. Immunoprecipitation of the insulin receptor was performed using 5 µg of the monoclonal insulin receptor antibody 29B4, as detailed previously (27).

Immunoblotting
For phosphotyrosine detection the immunoprecipitates were separated by SDS-PAGE using gradient (8–18%) horizontal gels and transferred to polyvinylidene difluoride filters in a semidry blotting apparatus (28). Filters were blocked for 60 min in PBS, pH 7.4, containing 1% BSA. Thereafter, filters were incubated for 16 h at 4 C with a 1:1000 dilution of the antiphosphotyrosine antibody conjugated to alkaline phosphatase. Substrates for alkaline phosphatase were then added for appropriate color development. For detection of the p85 regulatory subunit of PI 3-kinase, the blots were incubated overnight with the p85 antiserum at a 1:1000 dilution. After extensive washing, filters were incubated for 2 h with [125I]protein A, washed, air-dried, and visualized on a Fujix BAS 1000 bioimaging analyzer (Fuji Photo Film Co., Ltd., Tokyo, Japan). Quantification was performed densitometrically using BioImage whole band analysis software (Millipore Corp., Eschborn, Germany). Immunoblotting of PPAR{gamma} was performed using whole cell extracts (lysis buffer: 10 mM Tris, 5 mM EGTA, 0.1 mM dithiothreitol, 2 mM phenylmethylsulfonylfluoride, 0.12 µg/ml leupeptin, and 0.5% SDS) from cardiomyocytes and human adipocytes (27). Blots were incubated with the PPAR{gamma} antiserum at a 1:2000 dilution as described above and processed for enhanced chemiluminescence detection using a horseradish peroxidase-conjugated goat antirabbit antibody.

Assay of PI 3-kinase activity
PI 3-kinase activity was measured directly in IRS-1 immunoprecipitates in 50 µl of a reaction mixture containing 0.2 mg/ml PI, 20 mM HEPES (pH 7.1), 0.4 mM EGTA, 0.4 mM Na2HPO4, and 10 mM MgCl2 in the absence or presence of wortmannin (1 µM). The latter completely blocks PI 3-kinase activity and was used for the determination of background activity. The kinase buffer was incubated with the immunoprecipitates for 5 min at room temperature, and the reaction was started by addition of [{gamma}-32P]ATP (40 µM and 0.1 µCi/µl). After 20 min, the reaction was stopped by the addition of 30 µl 4 N HCl and 130 µl chloroform-methanol (1:1). The organic phase was extracted and spotted on a silica gel TLC plate (Merck) and developed in chloroform-methanol-25% NH4OH-water (43:38:5:7, vol/vol/vol/vol). Plates were dried and subsequently visualized and analyzed on a Fujix BAS 1000 bioimaging analyzer.

Presentation of data and statistics
All data analysis was performed using Prism (GraphPad, San Diego, CA) or t-ease (ISI, Philadelphia, PA) statistical software. The significance of reported differences was evaluated using the null hypothesis and t statistics for paired data. Corresponding significance levels are indicated in the figures.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of MCC-555 on glucose transport
To gain initial insights into the action profile of MCC-555 in cardiomyocytes, we preincubated the cells with two different concentrations of the drug (10 and 100 µM, respectively) for 30 min followed by determination of the initial influx (10 sec) of 3-OMG, representing carrier-mediated transport. As shown in Fig. 2Go (upper panel, left), MCC-555 did not modify basal glucose transport, suggesting that this compound does not elicit insulin-like signals in an acute fashion. On the other hand, the stimulatory action of insulin on glucose transport was markedly enhanced in the presence of high concentrations of MCC-555. Thus, the incremental increase in glucose transport due to insulin at physiological concentrations of the hormone (3 x 10-11 M) was enhanced about 3-fold from 169 to 476 pmol 3-OMG/106 cells·10 sec (Fig. 2Go, upper panel, right). A significant, but less pronounced, effect was also detected for the maximum insulin response (increase of 1.7-fold over the control value). This acute response of MCC-555 was only detected at a 100-µM concentration of the drug. We therefore extended the incubation periods using a 2-h protocol and analyzed the effects of 10 µM MCC-555 on glucose transport in the absence and presence of physiological concentrations of insulin (Fig. 2Go, lower panel, left). Again, no significant effect of the drug on basal glucose uptake was detected, supporting our above-mentioned conclusions. However, MCC-555 significantly enhanced the stimulatory action of the hormone. Under these conditions, the incremental increase in glucose transport was enhanced 2.8-fold from 125 to 348 pmol 3-OMG/106 cells·10 sec (Fig. 2Go, lower panel, right). These data show that low concentrations of the drug are able to produce a marked sensitization of insulin action in this cellular system. The requirement of longer incubation periods at 10 µM may be indicative of a slow uptake process of the drug and/or the requirement of intracellular metabolism to achieve drug action. It is worth noting that other TZD, such as troglitazone, were unable to produce acute sensitization of insulin action in cardiomyocytes even at high concentrations (50 µM) applied to the cells for 3 h (9). This may be indicative of potential differences between MCC-555 and other TZD concerning molecular action mechanisms, at least in this cellular system.



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  		Figure 2. Effect of MCC-555 on basal and insulin-stimulated glucose transport. Freshly isolated cardiomyocytes (4 x 105 cells/ml) were preincubated for 30 min (upper panel) or 2 h (lower panel) with the indicated concentrations of MCC-555. Cells were then stimulated with insulin for 5 min, and the transport of 3-OMG (final concentration, 100 µmol/l) was determined over a 10-sec assay period, as outlined in Materials and Methods. Data are presented as a percentage of basal transport rates (left panels) or as absolute transport rates (right panels) and are the mean ± SEM of three to six separate experiments. *, Significantly different from corresponding control at P < 0.005; **, P < 0.05.

 
Studies on insulin signaling
The data reported to date suggested that MCC-555 could be able to modify certain elements of the insulin signaling cascade, possibly by enhancing insulin-induced protein tyrosine phosphorylations and/or modifications of downstream signaling. We therefore analyzed the tyrosine phosphorylation of the insulin receptor (IR) ß-subunit and of IRS-1 in response to insulin (100 nM) using the 30-min preincubation protocol with 100 µM MCC-555 outlined in Fig. 2Go. A representative Western blot of the tyrosine-phosphorylated IR ß-subunit is presented in Fig. 3Go (upper panel). Insulin produced a marked increase (3.3 ± 0.2-fold; n = 3) in the autophosphorylation of the IR, with a slight, but insignificant (P > 0.05), modification in the presence of MCC-555 (3.8 ± 0.2-fold; n = 3). When MCC-555 was applied in the absence of insulin, no autophosphorylation of the IR became detectable (Fig. 3Go, upper panel). Comparable results were obtained at lower insulin concentrations (0.1 nM; data not shown).



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  		Figure 3. Insulin-activated tyrosine phosphorylation of IR and IRS-1 in the absence or presence of MCC-555. Cardiomyocytes were incubated in the absence or presence of 100 µM MCC-555 for 30 min and subsequently stimulated with insulin (10-7 or 10-10 M) for 5 min. After lysis, either IR (upper panel) or IRS-1 (lower panel) were immunoprecipitated, immune complexes were solubilized in Laemmli buffer, and submitted to separation by SDS-PAGE on gradient gels. Immunoblotting was then performed using an antiphosphotyrosine antibody coupled to alkaline phosphatase, as outlined in Materials and Methods. Substrate was added for appropriate color development, and visualized signals were quantified using BioImage whole band analysis software. Representative experiments of three performed are shown.

 
Phosphotyrosine analysis of IRS-1 immunoprecipitates from cardiomyocytes indicated the presence of a Tyr-phosphorylated protein with a molecular mass between 160–170 kDa (Fig. 3Go, lower panel), previously identified as IRS-1 in these cells (20). Tyr phosphorylation of IRS-1 was increased in response to insulin to the same level in both the absence and presence of MCC-555 (151 ± 7% vs. 158 ± 18% of the control value, respectively; P > 0.05; n = 3). Comparable results were obtained at higher insulin concentrations (data not shown).

It has now been recognized that PI 3-kinase represents an essential element for downstream insulin signaling to the glucose transporter GLUT4 in all major target tissues (29). We therefore investigated the effect of MCC-555 on the IRS-1-associated PI 3-kinase activity in cardiomyocytes stimulated with physiological insulin concentrations (Fig. 4Go). A representative autoradiogram of the TLC of the PI 3-kinase products using phosphatidylinositol as a substrate is shown in Fig. 4Go (upper panel). As can be clearly seen from the data, the insulin-induced enzyme activity is strongly enhanced by preincubating the cells with the drug. Quantification of three separate experiments (Fig. 4Go, middle panel) indicated a more than 2-fold higher IRS-1-associated PI 3-kinase activity in the presence of 10 µM MCC-555. The same potentiation of insulin-stimulated PI 3-kinase activity was observed at 100 nM insulin (Fig. 4Go, middle panel). The effect of MCC-555 was dose dependent; it was maximal at 10 µM using the 2-h protocol (Fig. 4Go, lower panel).



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  		Figure 4. Effects of MCC-555 on IRS-1-associated PI 3-kinase activity. Upper panel, Cardiomyocytes were incubated for 2 h at 37 C with 100 µM MCC-555. Insulin (10-10 M) was then added to each sample for 5 min. After solubilization, IRS-1 was immunoprecipitated as described in Materials and Methods. The PI 3-kinase assay was then performed in the immunoprecipitate using [32P]ATP and PI as substrate. Lipid products were separated by TLC, and signals were visualized on a Fujix BAS 1000 BioImaging analyzer. Middle panel, Quantification of autoradiographs was performed using BioImaging analysis software. Results are expressed as a fold over basal PI 3-kinase activity measured in the absence of MCC-555 and insulin. All data were corrected for enzyme activity not inhibited by wortmannin. The results shown are the mean ± SEM of three or four separate experiments. Lower panel, Cardiomyocytes were preincubated with increasing concentrations of MCC-555 for 2 h, stimulated with insulin for 5 min, and processed for PI 3-kinase determination as outlined above. Data are the mean ± SEM of three separate experiments.

 
The augmented insulin-stimulated PI 3-kinase activity in the presence of MCC-555 could not be explained by an increased association of the p85 regulatory subunit with IRS-1, as shown in the representative Western blot in Fig. 5Go. Insulin markedly stimulated the coimmunoprecipitation of p85 and IRS-1; however, in four separate experiments only a slightly and insignificantly higher association of p85 to IRS-1 was detected in cardiomyocytes preincubated with MCC-555 (100 µM) for 2 h. Total expression of p85 also remained unaffected (data not shown). As PI 3-kinase activity was increased at least 2-fold by the drug (see Fig. 4Go), it can be concluded that MCC-555 modulates the intrinsic activity of PI 3-kinase associated with IRS-1 without affecting recruitment of the enzyme to the docking protein.



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  		Figure 5. Association of p85 subunit of PI 3-kinase to IRS-1 in response to insulin in the absence or presence of MCC-555. After preincubation with MCC-555 (100 µM; for 2 h), the cells were stimulated with insulin (10-7 M) for 5 min. IRS-1 was immunoprecipitated as described in Fig. 3Go. The immunopellet was separated by SDS-PAGE and immunoblotted with a polyclonal anti-p85 antibody. Filters were incubated with [125I]protein A, and signals were visualized on a phosphorimager. One representative experiment of four is shown.

 
Involvement of protein synthesis in the action of MCC-555
As outlined above, the transcription factor PPAR{gamma} is considered to represent a major target for the intracellular action of TZD. This protein is expressed at a high level in adipose tissue (17), but more recent studies have also reported expression of PPAR{gamma} in heart and skeletal muscle (30). Using human adipocytes as a reference tissue, we have now performed Western blotting analysis of PPAR{gamma} to confirm the expression of this protein in our cardiomyocyte preparation. As shown in Fig. 6Go, the PPAR{gamma} antiserum recognized a protein band with a molecular mass of about 53–58 kDa in adipocytes and cardiomyocytes. The abundance of PPAR{gamma} in cardiocytes can be roughly estimated to be 20–30% of that in adipocytes (Fig. 6Go).



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  		Figure 6. Western blot analysis of PPAR{gamma} expression. Whole cell extracts were prepared from human adipocytes (Adi) or rat cardiomyocytes (Cardi) as outlined in Materials and Methods. Proteins were separated by SDS-PAGE and immunoblotted with a polyclonal PPAR{gamma} antibody. Filters were then incubated with horseradish peroxidase-conjugated goat antirabbit antibody and processed for ECL detection. Four experiments with similar results were performed.

 
To assess whether the acute effects of MCC-555 on insulin-stimulated glucose transport might involve protein synthesis and thus most probably the PPAR{gamma} pathway, we preincubated the cardiomyocytes with cycloheximide before performing the 30-min incubation protocol with MCC-555. As presented in Fig. 7Go and as expected, cycloheximide was essentially unable to alter the insulin-stimulated glucose transport rate. However, potentiation of insulin action by MCC-555 was completely abolished by inhibition of protein synthesis (Fig. 7Go). These findings suggest the existence of a very rapidly induced protein that modulates insulin action at the level of PI 3-kinase. It should be noted that initial experiments revealed a 50-fold lower affinity of MCC-555 for PPAR{gamma} compared with the high affinity TZD ligand BRL 49653 (31) despite a more potent in vivo action profile of MCC-555. Most interestingly, it was observed that MCC-555 is able to function as a context-dependent antagonist or partial agonist for PPAR{gamma}, making it unique among TZDs (31).



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  		Figure 7. Cycloheximide blocks the potentiation of insulin action by MCC-555. Cardiomyocytes were subjected to the indicated incubation conditions using insulin (10-7 M) for 5 min and MCC-555 (100 µM) and cycloheximide (1 mM) for 30 min. Initial rates of 3-OMG transport were then determined, as outlined in Fig. 2Go. Glucose transport is expressed as a percentage of basal uptake, which was determined in the absence of any addition. Data are the mean ± SEM (n = 3–4). *, Significantly different from transport stimulation determined in the presence of cycloheximide (P = 0.0187).

 
Effects of MCC-555 in cardiomyocytes from obese Zucker rats
The data reported above highlight the strong insulin-sensitizing potency of MCC-555 at the level of glucose transport and insulin signaling. To evaluate whether this drug action is able to overcome insulin resistance, we isolated cardiomyocytes from genetically (fa/fa) obese Zucker rats. We recently reported (20) a largely blunted half-maximal response of glucose transport in these cells with a marked rightward shift of the dose-response curve for insulin. This was explained by hyperphosphorylation of IRS-1 on Ser/Thr and a defective activation of PI 3-kinase. In light of these findings we investigated whether MCC-555 was able to restore the normal response of the glucose transporter using a short term preincubation of cardiomyocytes isolated from obese Zucker rats. As presented in Fig. 8Go (upper panel), these cells are completely insulin resistant at 0.03 nM insulin, whereas a 1.5- to 2-fold response is normally observed in control cells at this hormone concentration (see also Fig. 2Go). Preincubation with MCC-555 completely ameliorated the insulin resistance and produced a substantial increase in insulin-mediated 3-OMG uptake from 26 to 219 pmol/106 cells·10 sec (Fig. 8Go, lower panel) at this low physiological concentration. At higher concentrations of the hormone, 3-OMG uptake was also significantly elevated from 300–513 pmol/106 cells·10 sec (Fig. 8Go, lower panel). This observation clearly indicates that MCC-555 is able to normalize and improve insulin action in a severe insulin-resistant state.



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  		Figure 8. MCC-555 ameliorates insulin resistance in cardiomyocytes from obese Zucker rats. Isolated cardiomyocytes from genetically obese (fa/fa) Zucker rats were preincubated with 100 µM MCC-555 for 30 min, followed by incubation with the indicated concentrations of insulin for 5 min. 3-OMG transport was then determined as outlined in Fig. 2Go. Data are presented as a percentage of basal transport rates (upper panel) or as absolute transport rates (lower panel) and are the mean ± SEM of three individual experiments, each performed in triplicate.

 
As outlined above, we reported that the hyperphosphorylation of IRS-1 could be the underlying cause for the defective activation of PI 3-kinase in myocytes from obese Zucker rats (20). We therefore investigated whether MCC-555 might be able to reduce this hyperphosphorylation of IRS-1. Cardiomyocytes were isolated from Zucker rats and labeled with [33P]orthophosphate for 2.5 h in the absence or presence of MCC-555. As shown in our earlier work (20), for IRS-1 this in vitro labeling approach can be used to provide a valid estimate of the Ser/Thr phosphorylation state. IRS-1 was then immunoprecipitated and analyzed by SDS-PAGE followed by autoradiography. The representative experiment shown in Fig. 9Go indicates that the drug induces a substantial reduction in the phosphorylation state of IRS-1. Quantification of three separate experiments showed that MCC-555 reduces the Ser/Thr phosphorylation of IRS-1 in cardiomyocytes from obese rats by about 50% (P = 0.002).



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  		Figure 9. Effect of MCC-555 on the phosphorylation state of IRS-1. Cardiomyocytes (1 x 106 cells/ml) from obese Zucker rats were labeled with [33P]orthophosphate for 2.5 h at 37 C, as described in Materials and Methods. Cells were subsequently lysed, and IRS-1 was immunoprecipitated and analyzed by SDS-PAGE and autoradiography. A representative experiment of three performed is shown.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
TZD compounds have been recognized to function as insulin sensitizers (1, 2), and the rapidly growing family of these drugs (24, 32) might be considered the most promising approach for novel therapy of different states of insulin resistance. To date, the vast majority of available data suggests that these compounds do not acutely modify the insulin signaling cascade and that long term incubations are required to produce the insulin-sensitizing effects (1, 2, 8, 9, 10, 16, 33, 34). MCC-555 is a novel TZD exhibiting a very high in vivo potency in rodent models of insulin resistance (24). A key finding of the present study consists in the observation that this TZD is able to acutely sensitize cardiac glucose uptake 2- to 3-fold at physiological insulin concentrations (3 x 10-11 M), without any modification of the basal transport rate. We recently reported completely different observations using another TZD, troglitazone, in that short term incubations of cardiomyocytes with the drug, even at very high concentrations, did not produce any effect in these cells (9). Instead, as consistently reported in cultured muscle and adipose cells, chronic exposure to TZD results in enhanced expression of glucose transporters GLUT1 and GLUT4, leading to a higher basal glucose uptake rate and possibly mediating the insulin-sensitizing effects of the drugs (9, 16, 33, 34, 35). Very recently, Wu et al. (36) reported that TZD activate GLUT4 transcription by PPAR{gamma} in differentiating 3T3 fibroblasts, either alone or in combination with C/EBPß and -{delta}, by a process requiring 24–48 h. We therefore conclude that the marked acute insulin-sensitizing potency of MCC-555 in cardiomyocytes involves a mechanism completely different from that of troglitazone, in agreement with our observations of a rapid concomitant modification of the insulin signaling cascade (see below) by MCC-555. Certainly, it may be anticipated that upon chronic exposure, this TZD may also enhance glucose transporter expression, and further work will be needed to explore this pathway, which could additionally contribute to the high in vivo potency of this drug.

Extensive investigations have been carried out to identify potential targets of TZD action upstream of GLUT4 at the level of the insulin signaling cascade. Thus, earlier work demonstrated that insulin receptor autophosphorylation and kinase activity could be normalized by pioglitazone treatment in fatty Zucker rats (37) and high fat-fed rats (38). Subsequent work showed that troglitazone was able to prevent the glucose-induced desensitization of the insulin receptor kinase, possibly involving the modulation of PKC (39). However, any direct effect of TZD on insulin binding and receptor tyrosine kinase activity has been excluded (21). Further, the insulin-stimulated tyrosine phosphorylation of IRS-1 remained unaffected following a 5-h preincubation of Chinese hamster ovary cells with different TZD (21). In complete agreement with these findings we were unable to see any modification of insulin-regulated tyrosine phosphorylation of cardiac IR and IRS-1 in response to MCC-555. We therefore conclude that these early elements of the insulin signaling cascade do not represent primary targets of TZD action. Instead, we show here a marked potentiation of insulin action on IRS-1-associated PI 3-kinase activity after short term preincubation of cardiomyocytes with MCC-555, being exactly coincident with the activation of glucose transport. The observed potentiation of insulin-stimulated PI 3-kinase principally agrees with earlier studies of Berger and co-workers (21, 22) using adipose tissue and myotubes; however, some specific differences need further consideration. First, the effects of TZD on PI 3-kinase in L6 myotubes have not been correlated to glucose transport and were not shown to be related to the IRS-1-associated PI 3-kinase activity (21). Second, in Chinese hamster ovary cells, the TZD increased the amount of p85 subunit of PI 3-kinase in antiphosphotyrosine immunoprecipitates after stimulation with insulin. This differs from our findings showing an unaltered abundance of p85 in IRS-1 immunoprecipitates in response to MCC-555 and insulin. It should also be noted that the effects of pioglitazone on insulin-stimulated PI 3-kinase activity could not be confirmed in 3T3-L1 adipocytes using both acute and long term protocols (23). Therefore, it remains to be elucidated whether PI 3-kinase represents a general target for all TZD or if certain structural requirements are implicated in the modulation of this key enzyme of insulin signaling. At least for MCC-555 we would like to conclude that the drug increases the intrinsic activity of IRS-1-associated PI 3-kinase, most likely explaining the sensitization of insulin-stimulated glucose transport. Changes in the intrinsic activity of PI 3-kinase are consistent with the unaltered tyrosine phosphorylation of IRS-1 and could involve the Ser phosphorylation of p85 by PI 3-kinase, a process known to inhibit the enzyme activity (40), or the Ser-phosphorylation of PI 3-kinase docking proteins such as IRS-1. Initial evidence to support this view stems from our observation that MCC-555 was indeed able to promote Ser/Thr dephosphorylation of IRS-1 in cardiac cells from obese rats (see below). Further work will be needed to clarify the precise molecular steps of PI 3-kinase modulation by this compound.

A member of the PPAR family of nuclear receptors, PPAR{gamma}, has been implicated in the control of adipocyte differentiation and expression of fat-specific genes (17) and must be considered as the major target for intracellular action of TZD (1, 2, 14, 15, 32). Expression of PPAR{gamma}2 appears to be restricted to adipose tissue (41), whereas PPAR{gamma}1 is found in various tissues, including heart and skeletal muscle in both rodents (42, 43) and humans (30, 41), albeit at a much lower level. Using RT-PCR, Auboeuf et al. (41) reported that PPAR{gamma} in skeletal muscle amounts to only 3–4% of the expression seen in fat. Interestingly, at least in rodents a considerably higher PPAR{gamma} expression has been reported for cardiac muscle (3- to 5-fold higher; estimated from Fig. 1Go of Ref. 41) compared with skeletal muscle (42, 44). In the present investigation we have determined the protein expression of PPAR{gamma} in our cardiomyocyte preparation and found it to be 20–30% of that in adipocytes, in excellent agreement with the above-mentioned investigations. This observation strongly suggests that the induction of glucose transporter expression by long term treatment of cardiomyocytes with troglitazone, as reported in our earlier study (9), results from activation of the cardiac PPAR{gamma} receptor. Most interestingly, we show here that inhibition of protein synthesis completely eliminates the rapid effects of MCC-555 on insulin signaling. Based on available binding data it is reasonable to assume that the action of MCC-555 involves the PPAR{gamma} receptor (31). The novel properties of MCC-555 with respect to PPAR{gamma} activation (31) may at least partly explain the differential response of cardiomyocytes observed with this TZD and troglitazone (9). Given the observation that MCC-555 enhances the intrinsic PI 3-kinase activity (see above), it is tempting to speculate that this drug controls the synthesis of a PI 3-kinase modulator protein. Such a hypothetical protein could be a PI 3-kinase activator, a phosphatase, phosphatase activator (see discussion below), or a kinase inhibitor. Identification of this protein and the elucidation of its physiological regulation might represent a major goal for future work.

In a recent report from this laboratory we showed that cardiac insulin resistance of obesity correlates to a reduced sensitivity of glucose transport, probably resulting from a defective activation of PI 3-kinase (20). We show here that a short preincubation of cardiomyocytes from obese Zucker rats with MCC-555 completely ameliorates the insulin resistance, leading to a normal half-maximal and increased maximal insulin response of glucose transport. These data suggest that the mechanisms of PI 3-kinase modulation by MCC-555, as discussed above, also operate in an insulin-resistant cell and enable the drug to correct the defect in insulin action. Correction of insulin signaling defects in fatty Zucker rats has also been reported for pioglitazone under in vivo conditions (45). However, this failed to normalize the defect in glucose transport and glucose transporter translocation (46), suggesting that this TZD does not modify the specific elements of insulin signaling leading to GLUT4 translocation (23). Cardiac insulin resistance of insulin signaling was paralleled by a hyperphosphorylation of IRS-1 on Ser/Thr, which is known to function as a negative control mechanism (20). This hyperphosphorylation was largely reduced in the presence of MCC-555. It may be speculated that this dephosphorylation is linked to the normalization of PI 3-kinase and glucose transport activation, although direct evidence for this relationship is still missing. Tumor necrosis factor-{alpha} is also known to induce insulin resistance by serine phosphorylation of IRS-1 in adipocytes (47). As recently shown by Peraldi et al. (48), the effect of tumor necrosis factor-{alpha} on insulin signaling can be essentially blocked by TZD, supporting our view that dephosphorylation of critical proteins on Ser/Thr may be a major step in the molecular pathways of TZD action.

In summary, MCC-555 is a highly potent new TZD that is able to potentiate insulin signaling by increasing the intrinsic activity of IRS-1-associated PI 3-kinase, leading to enhanced glucose transport in normal and insulin-resistant cardiomyocytes. This drug effect is dependent on protein synthesis and may involve the dephosphorylation of signaling intermediates such as IRS-1. It is suggested that MCC-555 provides a causal therapy of insulin resistance by targeted action on the defective site.


   Footnotes
 
1 This work was supported by the Ministerium für Wissenschaft und Forschung des Landes Nordrhein-Westfalen, the Bundesministerium für Gesundheit, EU COST Action B5, and a grant from Mitsubishi Chemical (Yokohama, Japan). Back

Received March 18, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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