Rosiglitazone Treatment in Zucker Diabetic Fatty Rats Is Associated With Ameliorated Cardiac Insulin Resistance and Protection From Ischemia/Reperfusion-Induced Myocardial Injury

This study was conducted in accordance with the guidelines for care and use of laboratory animals of the U.S. National Institutes of Health. Male ZDF rats (ZDF/Gmi-fa/fa) and lean Zucker (fa/?) rats (Genetic Models) at age 12–14 weeks were used (n = 245). Unless otherwise indicated, rosiglitazone maleate (GlaxoSmithKline) was administered at 3 mg · kg⁻¹ · day⁻¹ via oral gavage for 7 days, and the eighth dose was given 1 h before ischemia. The animals were premedicated with atropine sulfate (0.04 mg/kg intramuscularly) and then anesthetized with Nembutal (60 mg/kg i.p.; Abbott Laboratories). Trachea were intubated and ventilated with a Harvard small animal respirator. The electrocardiogram and the body temperature of the rats were monitored throughout the experiment. Coronary artery (left anterior descending artery) occlusion (30 min) and reperfusion (4 h for apoptosis and 24 h for infarct and functional assessment) were induced by inflating and then deflating a nontraumatic balloon occluder that was fixed on the left anterior descending artery. The successful performance of coronary occlusion and reperfusion was verified by visual inspection of color in the apex and typical electrocardiogram changes.

In a separate group of rosiglitazone- and vehicle-treated ZDF rats, indwelling carotid artery and jugular vein catheters were surgically implanted. Upon recovery from surgery, rats were fasted overnight before baseline or normoglycemic-hyperinsulinemic cardiac glucose oxidation measurements. A normoglycemic-hyperinsulinemic (10 mU · kg⁻¹ · min⁻¹, Humulin Regular; Eli Lilly, Indianapolis, IN) clamp was initiated in awake rats by administering a variable rate [1-¹³C]glucose (99% enriched, 20% wt/vol; Cambridge Isotope Laboratories, Cambridge, MA) infusion at 2.5 min after the commencement of a primed (150 mU · kg⁻¹ · min⁻¹ for 0–45 s, 75 mU · kg⁻¹ · min⁻¹ for 45–90 s) continuous insulin infusion. Plasma glucose concentrations were clamped at normoglycemic levels for 75 min. Blood samples were drawn at baseline, 7.5 min, 15 min, and every 15 min thereafter for immediate assessment of plasma glucose and lactate concentrations (2300 STAT Plus analyzer; YSI, Yellow Springs, OH). Baseline measurements were performed in awake rats by maintaining a [1-¹³C]glucose (10 mg · kg⁻¹ · min⁻¹)/somatostatin (1.5 μg/min) infusion for 120 min. At the end of the clamp experiments, rats were anesthetized with Nembutal at 50 mg/kg i.v., and the heart was rapidly removed and frozen in liquid nitrogen.

Cardiac tissue extracts were prepared for high-field nuclear magnetic resonance (NMR) analysis by homogenizing ∼0.3 g of the heart in a similar fashion as previously described (25). NMR analysis was performed at 9.4 Tesla (WB-400 Avance NMR spectrometer; Bruker Medical, Billerica, MA). Proton-observed, carbon-enhanced spectroscopy was performed on tissue extract samples for measuring [3-¹³C]lactate, [3-¹³C]alanine, and [4-¹³C]glutamate fractional enrichments (25). The relative cardiac glucose and fat/ketone oxidation rates in terms of acetyl-CoA units were assessed from the metabolite pool enrichments. The degree to which the [4-¹³C]glutamate pool was diluted from the [3-¹³C]pyruvate (equivalent to the [3-¹³C]lactate and [3-¹³C]alanine pool enrichment) pool depends on the extent of label dilution from unlabeled fat oxidation present. Therefore, the relative glucose oxidation (%) is defined as {[4-¹³C]glutamate/[([3-¹³C]lactate + [3-¹³C]alanine)/2]} × 100, and relative fat/ketone oxidation (%) is calculated as (1 − relative glucose oxidation) × 100.

Myocardial Akt, phospho-Akt (Akt-pS473), phospho-FKHR (forkhead transcription factor; FKHR-pS256), and phospho-glycogen synthase kinase (GSK)-3β (GSK-3β-pS9) in the ischemic myocardium after 4-h reperfusion were determined by Western blot analysis as reported previously (26). Briefly, protein extracts (50–100 μg) from ischemic myocardium collected at 4 h after reperfusion were fractionated on 10% polyacrylamide gel under reducing conditions, transferred onto nitrocellulose membranes, and probed with the following primary antibodies against Akt: Akt-pS473, FKHR-pS256, and GSK-3β-pS9 (all antibodies from Cell Signaling). Blots were incubated with horseradish peroxidase–conjugated second antibody, and the signal was detected with LumiGlo chemiluminescent reagent (Cell Signaling). β-Actin was used for normalization.

Ischemic myocardial tissues collected 4 h after reperfusion were lysed and immunoprecipitated with anti-Akt antibody and then used to measure Akt kinase activity using an Akt kinase assay kit (Cell Signaling) with GSK-3α/β as an exogenous substrate, according to the manufacture’s instructions.

The apoptotic myocytes in ischemic myocardium after 4-h reperfusion were determined by terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling (TUNEL) assay with a cell death detection kit (Roche), as described previously (26). Briefly, the tissue sections after permeability treatment were incubated with TUNEL reaction mixture containing terminal deoxynucleotidyl transferase and fluorescein-dUTP (deoxyuridine triphosphate). For double labeling, the tissue sections after incubation with TUNEL reaction mixture and blockade of endogenous peroxidase, and nonspecific binding sites were incubated with the monoclonal antibody against α-sarcomeric actin (Sigma). After washing, the slides were incubated with biotinylated secondary antibody, and the cardiomyocytes were finally visualized with Texas red avidin. For detection of total nuclei, the slides were covered with the mounting medium containing 4′,6-diamidino-2-phenylindole (DAPI). At least six slides per block were evaluated. For each slide, 15 fields were randomly chosen, and a total of 2.5–3 × 10³ myocytes per slide were counted. We determined the index of apoptosis, i.e., the number of TUNEL-positive myocytes divided by the total myocytes that were stained with anti–α-sarcomeric actin (see Fig. 4C) per field.

DNA ladder experiments were performed as described previously (27). In brief, ischemic myocardial tissues collected at 4 h after reperfusion were minced in lysis buffer on ice, and proteinase K (100 μg/ml) was added. After incubation at 55°C with shaking for 18 h, DNA was extracted with phenol/chloroform three times, precipitated in ethanol, treated with DNA-free RNase, re-extracted, and precipitated again. DNA concentration was determined, and 10 μg of DNA was used for electrophoreses on a 1.8% agarose gel.

The ischemic area (area at risk) was distinguished from the area not at risk by Evans blue dye staining, and the infarcted portion of the myocardium (necrotic area) was determined by the triphenyl tetrazolium chloride method as described in detail previously (20). The three portions (i.e., area not at risk, area at risk, and necrotic area) of the left ventricle were quantified by use of Image-Pro software.

LV pressure and the arterial blood pressure were measured via two 1.4 F Millar Mikrotip catheter transducers that were inserted into the LV cavity through the right carotid artery and the right femoral artery, respectively. LV pressure and arterial blood pressure were digitally processed via a hemodynamic analyzing system (Gould 3P 6600). Mean arterial blood pressure, LV systolic pressure, LV end diastolic pressure, positive and negative maximal values of the first derivative of LV pressure (+dp/dt and -dp/dt), and heart rate were derived by computer algorithms (20).

Data are expressed as the means ± SE and analyzed by one-way ANOVA with subsequent post hoc paired comparisons or by unpaired Student’s t test. Differences with a value of P < 0.05 were considered statistically significant.

As shown in Table 1, treatment with rosiglitazone for 8 days significantly reduced the circulating levels of glucose, triglycerides, and free fatty acids in ZDF rats. During a normoglycemic-hyperinsulinemic clamp, the 8-day rosiglitazone–treated animals exhibited an increased insulin-stimulated glucose infusion rate from 13.2 ± 1.4 to 19.1 ± 2.4 mg · kg⁻¹ · min⁻¹ for vehicle and rosiglitazone, respectively (P < 0.03, n = 8). NMR experiments revealed that glucose oxidation under baseline conditions represented a small fraction of the entire substrate oxidation in the heart, and 8-day treatment with rosiglitazone had no effect on this measure (Fig. 1). After insulin stimulation, both 8-day rosiglitazone and vehicle groups exhibited an increase in relative cardiac glucose oxidation. However, the rosiglitazone-treated animals exhibited a markedly increased insulin-sensitized glucose oxidation rate versus vehicle-treated animals (Fig. 1). Conversely, fat/ketone oxidation in the heart was lower in rosiglitazone-treated animals versus the vehicle group (41.2 ± 4.8 vs. 54.7 ± 0.9% acetyl-CoA source, respectively; P < 0.02; n = 8).

As shown in Fig. 2, treatment with rosiglitazone for 8 days resulted in a significant increase in Akt phosphorylation (Akt-pS473) in the ischemic myocardium (Fig. 2A). Quantitative measurement by Western blot analysis showed a 2.1-fold increase in Akt-pS473 in the rosiglitazone-treated group versus the vehicle group (P < 0.01, n = 8) (Fig. 2B). The result of the Akt kinase assay (Fig. 2C) was consistent with the Western blot analysis, showing a 1.8-fold increase in cardiac Akt activity in rosiglitazone-treated animals compared with the vehicle group (P < 0.01, n = 8). There was no difference in myocardial levels of total Akt between the rosiglitazone-treated and the vehicle groups (Fig. 2A).

GSK-3 and FKHR are two downstream targets of Akt. As shown in Fig. 3B and C, treatment with rosiglitazone enhanced the phosphorylation of GSK-3 (GSK-3β-pS9) by 2-fold (P < 0.05, n = 8) and FKHR (FKHR-pS256) by 2.9-fold (P < 0.01, n = 8) in the ischemic myocardium compared with the vehicle group, respectively. Figure 3A is a representative Western blot from one of two experiments showing similar results.

Myocardial tissue from sham-operated ZDF rats exhibited no DNA ladder formation (Fig. 4A, lane 2), but clear DNA ladder formation was observed in myocardial tissue from the hearts subjected to ischemia/reperfusion and receiving vehicle (Fig. 4A, lanes 3–8). In contrast, DNA ladder formation was less clear or almost not detectable in the ischemic myocardium from six ZDF rats treated with rosiglitazone (Fig. 4A, lane 9–14).

In addition, the TUNEL assay demonstrated that TUNEL-positive-staining myocytes (apoptotic myocytes) in ischemic myocardium reached 21.3 ± 3.5% in the vehicle-treated ZDF rats (Fig. 4B). The TUNEL-positive myocytes were mainly localized in salvaged areas surrounding the infarcted tissues (data not shown). The number of TUNEL-positive myocytes in the ischemic regions from rosiglitazone-treated ZDF rats was reduced to 8.9 ± 2.5% (n = 6, P < 0.01 vs. vehicle) (Fig. 4B). Under the same conditions (i.e., 30-min ischemia followed by 4-h reperfusion), the number of TUNEL-positive myocytes in the ischemic myocardium from vehicle-treated lean Zucker rats was significantly lower (14.3 ± 2.1%) compared with the vehicle-treated ZDF rats (P < 0.01, n = 6), and this number was reduced to 8.1 ± 1.2% in rosiglitazone-treated lean rats (P < 0.05 vs. lean + vehicle) (Fig. 4B). Figure 4C is representative photomicrographs of in situ detection of apoptotic myocytes (TUNEL positive) in the heart tissue from ZDF rats subjected to 30 min of ischemia and 4 h of reperfusion and treated with vehicle (column V, rows 1–3) or rosiglitazone (column R, rows 1–3) for 8 days, or sham operated (column S, rows 1–3).

There was no difference in ischemic area (area at risk) expressed as percent of left ventricle (Fig. 5A, left side), indicating a comparable degree of ischemic insult between vehicle- and rosiglitazone-treated groups after left anterior descending artery occlusion in ZDF rats. Treatment with rosiglitazone for 8 days reduced ischemia/reperfusion-induced cardiac infarct size (% ischemic area) by 45.5%, from 60.8 ± 2.5% (vehicle) to 33.1 ± 3.7.0% (rosiglitazone) (n = 10, P < 0.01) (Fig. 5A, right side).

Ischemia/reperfusion-induced myocardial infarct size in the lean Zucker rats was significantly smaller than the infarct size in the ZDF rats (P < 0.01) (Fig. 5A, right side), although the ischemic area (% left ventricle) was similar between the two groups (Fig. 5, left side). In rosiglitazone-treated lean rats, the ischemia/reperfusion-induced cardiac infarction was reduced by 26.5% compared with the vehicle group (P < 0.05, n = 8).

Treatment with rosiglitazone for 8 days significantly improved cardiac functional contraction in ZDF rats at 24 h after reperfusion, as shown in Fig. 5B. The values of LV systolic pressure, +dP/dt, and −dP/dt were significantly enhanced in the rosiglitazone-treated group compared with the vehicle group (all P < 0.05, n = 9). Additionally, the mean arterial blood pressure was also improved in the rosiglitazone-treated group versus the vehicle-treated group, but no difference in the heart rate between the two groups was observed (data not shown).

As shown in Fig. 6, pretreatment of the ZDF rats with wortmannin, a phosphatidylinositol (PI) 3-kinase inhibitor, 15 min before reperfusion (26) markedly attenuated the cardioprotective effect of 8-day rosiglitazone treatment (>90% reduction). However, wortmannin alone had no effect on ischemia/reperfusion-induced myocardial infarction.

To further differentiate the non–insulin-related cardioprotective effect of rosiglitazone, a separate group of ZDF rats were treated with rosiglitazone at 3 mg · kg⁻¹ · day⁻¹ for 2 days. There was no difference in glucose infusion rates between the rosiglitazone-treated and vehicle-treated groups (15.5 ± 1.2 and 15.4 ± 0.6 mg · kg⁻¹ · min⁻¹, respectively) during a normoglycemic-hyperinsulinemic clamp (n = 10) (Fig. 7A), indicating that 2-day rosiglitazone treatment did not change insulin resistance in the rats. The ischemia/reperfusion-induced cardiac infarct size (% ischemic area) was reduced by 22.1%, from 58.6 ± 1.8% (vehicle) to 46.2 ± 1.7% (rosiglitazone; n = 9, P < 0.05) (Fig. 7B). This reduction was significantly less when compared with the 8-day rosiglitazone-treated group (P < 0.05). Additionally, there was no activation in myocardial level of Akt-pS473 in rosiglitazone-treated animals, as shown in Fig. 7C. The apoptotic myocytes in the ischemic myocardium of ZDF rats was 16.7 ± 5.6% (n = 4, reduced by 21.6% compared with the vehicle group).

Treatment with rosiglitazone for 8 days in ZDF rats resulted in a decrease in the in vivo ischemia/reperfusion-induced cardiac infarct size by 46% and apoptosis by 58%. This cardiac protection was associated with improved LV function as well as increased cardiac insulin sensitivity. There was a significant increase in insulin-sensitized cardiac glucose oxidation and a reduction in fat/ketone oxidation in rosiglitazone-treated ZDF rats versus the vehicle group. Concomitantly, a significantly enhanced level of Akt signaling in myocardium was observed in rosiglitazone-treated animals.

In ZDF rats, GLUT4 protein expression is decreased and insulin-stimulated cardiac glucose uptake is impaired (10,23). Previous studies have argued for a potential role of glucose in cardioprotection from ischemic injury (28–30). It was shown that cardiac myocytes in the presence of glucose and glycolytic inhibitors such as 2-deoxyglucose or iodoacetate reversed the protective effect of glucose on hypoxic injury (28). In isolated rat hearts, cardiac output after ischemia/reperfusion was increased when glucose was added to lactate in the absence of insulin (29). Conversely, if glucose is absent in the presence of hypoxia for 30 min followed by 60 min of reperfusion, LV function is impaired to a greater extent (30). A recent in vitro study has demonstrated that rosiglitazone treatment results in the normalization of insulin-stimulated glucose uptake in isolated perfused obese Zucker rat hearts (23). As a result, the increased insulin-sensitized cardiac glucose oxidation observed in the present study may in part contribute to the cardioprotection by rosiglitazone in the ZDF rats. However, it is also known that ischemia/reperfusion results in an increase in free fatty acid levels, which aggravates the severity of myocardial infarction (31). Therefore, the cardioprotection observed after the 8-day rosiglitazone treatment in the present study might be indirectly mediated by lowering systemic free fatty acid levels.

Akt signaling has been widely recognized as an important downstream modulator of the insulin receptor, and it plays a critical role in maintaining insulin sensitivity in humans (32). Akt2-deficient mice show resistance to insulin’s effects on glucose metabolism and develop frank diabetes (33,34). A recent study reported that a mutation in the gene encoding the Akt2 in a family resulted in severe insulin resistance and diabetes, providing an example of a monogenic inherited defect in postreceptor insulin signaling that leads to human insulin resistance and diabetes (32). An increasing body of evidence also suggests that Akt signaling plays an important cytoprotective role against ischemic injury in the heart. In cultured rat neonatal cardiomyocytes, activation of Akt significantly reduced H₂O₂-induced apoptosis, and blockade of Akt signaling by PI 3-kinase inhibitor or overexpression of a dominant-negative mutant of PI 3-kinase abolished the protective effect of insulin (35). In isolated perfused hearts or in vivo rat studies, insulin administration reduced ischemia/reperfusion-induced infarct size, and this protective effect was abolished if the upstream or downstream signaling in the Akt pathway was inhibited by pharmacological inhibitor, suggesting that the cardioprotective effect of insulin is mainly mediated via Akt signaling (26). It has been reported that the cytoprotective effect of Akt signaling is attributable to the inhibition of GSK-3 and FKHR, two downstream targets of Akt known to be involved in cell death. GSK-3 is inactivated by Akt via phosphorylation at serine 9 or 21 (36). Both the in vitro and in vivo studies have demonstrated that inactivation of GSK-3 enhanced cell survival (37,38), improved postischemic cardiac function, and reduced infarct size (39). Phosphorylation of FKHR by Akt keeps this transcriptional factor retention in the cytoplasm and away from the nucleus, where it triggers activation of genes critical for inducing cell death (40). The present study demonstrated a significant increase in the levels of GSK-3β-pS9 and FKHR-pS256 in the ischemic myocardium from rosiglitazone-treated ZDF rats, further suggesting a role of the Akt signaling pathway in PPAR-γ–mediated cardioprotection. It is interesting to note in the current study that treatment with rosiglitazone did not change the total Akt level in the heart, but only the active form of Akt (Fig. 2A), suggesting that PPAR-γ–mediated activation of Akt may serve as a protective mechanism when the heart is under an insulting stress.

Previous studies have shown that PPAR-γ agonists protected the heart against ischemia/reperfusion injury in isolated perfused hearts (24) as well as in vivo in the nondiabetic pig (19) and rat (20–22). The enhanced insulin sensitivity by chronic treatment with troglitazone was found in nondiabetic pigs, and it was assumed to contribute to the improved recovery of LV function after cardiac ischemia (19). However, other actions of the PPAR-γ agonist that do not appear to be linked to insulin sensitization, such as anti-inflammatory activities (20,21,22) and inhibition of c-Jun NH₂-terminal kinase/activating protein 1 pathway (24), have been suggested as potential mechanisms responsible for PPAR-γ–mediated cardioprotection.

Nevertheless, the present study provides several lines of evidence suggesting a potential association between cardiac insulin resistance and enhanced susceptibility to ischemic insult. First, the ischemia/reperfusion-induced myocardial injury (apoptosis and infarction) was greater in the ZDF rats than in the lean controls, suggesting a role for insulin resistance in the enhanced myocardial susceptibility to ischemia. Second, a greater degree of cardioprotection by rosiglitazone was observed in ZDF versus lean controls, suggesting that the improved cardiac insulin sensitivity provided the additional degree of cardioprotection. This finding is consistent with our previous study in nondiabetic Lewis rats (20). The cardioprotection by rosiglitazone against ischemia/reperfusion injury in Lewis rats was significant (24% reduction versus vehicle) but much less compared with that observed in ZDF rats in the present study (46%) while the animals were treated with the same dose regimen (3 mg · kg⁻¹ · day⁻¹ orally for 8 days) (20).

To further differentiate the insulin-sensitizing from the non–insulin-sensitizing mechanism in the cardioprotective role of rosiglitazone, a 2-day rosiglitazone dosing regimen was also used in the present study. The lack of insulin sensitization in the ZDF rats under this 2-day dosing regimen was associated with significantly less cardioprotection than that observed after an 8-day rosiglitazone treatment. Nevertheless, approximately half of the protection seen after an 8-day rosiglitazone treatment was observed after only a 2-day rosiglitazone treatment. Finally, pretreatment of ZDF rats with wortmannin at a dose that completely blocked Akt phosphorylation and activation by insulin, as demonstrated previously (26), remarkably diminished but did not abolish the cardioprotective effect of rosiglitazone. These results suggest that although non–insulin-related mechanisms appear to be involved in the cardioprotection by rosiglitazone, the improvement in insulin sensitization appears to be associated with enhanced cardioprotection against ischemia/reperfusion-induced injury in rosiglitazone-treated ZDF rats. It is not clear at present to what extent insulin- and non–insulin-related mechanisms play a role in the cardioprotection observed in the diabetic and nondiabetic condition. Further studies are necessary to clearly define and differentiate the direct role for both insulin- and non–insulin-related cardioprotective mechanisms.

In conclusion, chronic treatment with rosiglitazone protects the heart from ischemia/reperfusion-induced cardiac injury and improves cardiac contractile dysfunction in ZDF rats. The improvement in cardiac insulin sensitivity after an 8-day rosiglitazone treatment, as reflected by increased Akt signaling and glucose oxidation, was associated with this cardioprotection.