INTRODUCTION

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IT IS WELL-ESTABLISHED that, under most physiological conditions, the expression of the glucose transporter isoform GLUT-4 is closely associated with the capacity for stimulation of skeletal muscle glucose transport activity by insulin (for recent comprehensive reviews see Refs. 12 and 18). For example, skeletal muscle with a high percentage of type I or IIa fibers expresses high levels of GLUT-4 protein and has a high capacity for insulin-stimulated glucose transport activity, whereas muscle with a predominance of type IIb fibers displays low levels of GLUT-4 protein and has a lower capacity for insulin-stimulated glucose transport activity (13, 21). Moreover, interventions, such as endurance exercise training, concomitantly enhance both the level of GLUT-4 protein and insulin action on glucose transport activity in skeletal muscle. This is true not only for muscle from normal, insulin-sensitive animals (22-24; see also reviews in Refs. 12 and 18), but also for muscle from insulin-resistant obese animal models, such as the obese Zucker rat (2, 5, 8, 26).

Our laboratory has recently demonstrated that chronic administration of the angiotensin-converting enzyme (ACE) inhibitor trandolapril, normally used as a blood-pressure-lowering intervention (4, 20), to the insulin-resistant obese Zucker rat results in parallel increases in GLUT-4 protein levels and in insulin-stimulated glucose transport activity in skeletal muscle (19, 26) and that these increases were completely additive to those induced by endurance exercise training (26). However, the question remains as to whether this modulation of skeletal muscle GLUT-4 protein levels and insulin-stimulated glucose transport activity by chronic ACE inhibition alone or in combination with exercise training is a general phenomenon applicable to conditions of normal insulin sensitivity or whether the additivity between the effects of ACE inhibition and exercise training is restricted to insulin-resistant skeletal muscle (26). Our laboratory has previously demonstrated that administration of trandolapril to lean Zucker (Fa/) rats, a model of normal insulin sensitivity, for 2 wk resulted in a small (15%) improvement in insulin-mediated skeletal muscle glucose transport activity, but this increase was not statistically significant (19).

In this context, the present study was designed to test the hypothesis that an interactive effect of exercise training and ACE inhibition on insulin action, as seen previously in insulin-resistant muscle from the obese Zucker rat (26), would also be found in the lean Zucker (Fa/) rat. Lean Zucker rats underwent either 6 wk of exercise training, 6 wk of treatment with the ACE inhibitor trandolapril, or 6 wk of combined exercise training and ACE inhibition. Subsequently, glucose tolerance, insulin action on muscle glucose transport activity, the muscle level of GLUT-4 protein, and the activities of enzymes involved in glucose phosphorylation (total hexokinase activity) and glucose oxidation (citrate synthase activity) were assessed.

	METHODS

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Animals. Female lean Zucker (Fa/?) rats were received from Harlan (Indianapolis, IN) at ~5 wk of age, weighing 130-140 g. The animals were housed in a temperature-controlled room (20-22°C) with a reversed 12:12-h light-dark cycle (lights on from 7 PM to 7 AM) at the Central Animal Facility of the University of Arizona. The animals had free access to chow (Purina, St. Louis, MO) and water. All procedures were approved by the University of Arizona Animal Use and Care Committee.

Oral glucose tolerance tests. After 6 wk, an oral glucose tolerance test (OGTT) was performed on each animal. The rats were restricted to 4 g of chow after 6 PM of the evening before the test. Between 8 AM and 9 AM on the day of the OGTT, ~12 h after the most recent trandolapril treatment and/or 24 h after the most recent exercise bout, the rats were administered a 1 g/kg body wt glucose load by gavage (7). Blood was collected from a small cut at the tip of the tail immediately before and at 15, 30, and 60 min after glucose administration. The animals were restrained in Plexiglas holding tubes for only as long as required to collect each blood sample, normally for only ~2-3 min for each sample. The whole blood was thoroughly mixed with EDTA (final concentration of 18 mg/ml) and centrifuged at 13,000 g to isolate the plasma. The plasma was stored at 80°C and subsequently assayed for glucose (Sigma Chemical, St. Louis, MO), insulin by radioimmunoassay (Linco, St. Louis, MO), and free fatty acids (Wako, Richmond, VA). After the final blood collection, each animal was given 2 ml of sterile saline subcutaneously to account for plasma volume loss. On completion of the OGTTs, animals in the exercise training groups were run for 30 min. No animals were treated with trandolapril on this day.

Peak oxygen consumption. Aerobic capacity was assessed in each animal by the peak rate of oxygen consumed (O_2 peak) during a treadmill test 48 h after the OGTT by using the method of Bedford et al. (3). No exercise was performed on the day before the O_2 peak tests; however, trandolapril was administered to the trandolapril group and to the combined exercise-trained and trandolapril-treated group. Animals were run on a motorized treadmill in an airtight Plexiglas chamber. Grade and speed of the treadmill were increased every 3 min from a basal level of 0% grade and 13.4 m/min through the following stages: 16.1 m/min at 5%, 21.4 m/min at 10%, 26.8 m/min at 10%, 32.2 m/min at 12%, 32.2 m/min at 15%, 32.2 m/min at 18%, and 32.2 m/min at 21%. The test was terminated when the rats were unable to keep pace with the treadmill belt. Oxygen (Ametek S-3A1, Applied Electrochemistry, Pittsburgh, PA) and carbon dioxide (Ametek CD-3A) were measured in expired gases every 3 min for the determination of oxygen uptake (ml O₂ · kg body wt¹ · min¹). Exercise training and trandolapril treatments were resumed the day after the O_2 peak assessment.

Muscle glucose transport activity. Approximately 72 h after the O_2 peak test and 24 h after the final exercise bout and/or 12 h after the final trandolapril treatment, animals were weighed and deeply anesthetized by using an intraperitoneal injection of pentobarbital sodium (50 mg/kg body wt). The determination of muscle glucose transport activity, assessed by using 2-deoxyglucose (2-DG) uptake, was started at 8 AM after an overnight food restriction, as described in Oral glucose tolerance tests. One soleus and both epitrochlearis muscles were dissected and prepared for in vitro incubation. Whereas the epitrochlearis muscles were incubated intact, the soleus muscle was prepared into two strips (~25 mg) and incubated. Each muscle was incubated for 1 h at 37°C in 3 ml oxygenated (95% O₂-5% CO₂) Krebs-Henseleit buffer (KHB) supplemented with 8 mM glucose, 32 mM mannitol, and 0.1% BSA (radioimmunoassay grade, Sigma Chemical). One epitrochlearis muscle and one soleus strip were incubated in the absence of insulin, and the contralateral epitrochlearis muscle and second soleus strip were incubated in the presence of a maximally effective concentration of insulin (2 mU/ml; Humulin, Eli Lilly, Indianapolis, IN).

Biochemical assays. The remaining two pieces of epitrochlearis (one from the muscle incubated in the absence of insulin and the other from the muscle incubated in the presence of insulin) were pooled together, reweighed, and homogenized in 40 volumes of ice-cold 20 mM HEPES (pH 7.4) containing 1 mM EDTA and 250 mM sucrose. These homogenates were used for the determination of total protein content by using the bicinchoninic acid method (Sigma Chemical), GLUT-4 protein level (14), total hexokinase activity (28), and citrate synthase activity (27). In addition, the contralateral soleus and plantaris muscles and the heart were removed, trimmed of fat and excess connective tissue, quickly frozen in liquid nitrogen, weighed, and used for subsequent determination of these same variables. As the protein concentration in these muscles was not altered by the interventions (data not shown), enzyme activities are expressed relative to muscle protein content.

Statistical analysis. All data are presented as means ± SE. The significant differences between groups was assessed by a factorial ANOVA with a post hoc Fisher's protected least significant difference test (StatView version 5.0, SAS Institute, Cary, NC). P < 0.05 was considered statistically significant.

	RESULTS

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Final body weights, muscle weights, O_2 peak, and maximum run times. Final average body weights for the exercise-trained group and combined exercise-trained and trandolapril-treated group were slightly, but significantly (P < 0.05), lower than those of the sedentary control group (Table 1). There were no significant differences among groups for average wet weights of the whole epitrochlearis, soleus, or plantaris muscles (data not shown). Trandolapril treatment alone induced a small, but statistically significant, reduction in heart mass relative to the sedentary control group, both in absolute (11%) and relative (6%) terms. Relative heart wet weight in the exercise-trained group was significantly larger (5%) than that in the sedentary control group. Interestingly, the reduced absolute heart mass of the trandolapril-treated group (11%) was still apparent when exercise training was combined with this intervention.

View this table: [in this window] [in a new window]   Table 2.   O2 peak and maximum run time to fatigue in lean Zucker rats after the 6-wk intervention periods

OGTT responses. Table 3 shows the fasting plasma levels of glucose, insulin, and free fatty acids in the experimental groups. Plasma glucose and insulin were not altered by the interventions. Compared with the sedentary control group, exercise training alone (18%) or in combination with trandolapril treatment (21%) resulted in a significant lowering of plasma free fatty acid levels. ACE inhibitor treatment did not significantly alter any of these variables in the lean Zucker rat.

View larger version (22K): [in this window] [in a new window]   Fig. 1.   Effects of chronic treatment with trandolapril, exercise training (Runners), or trandolapril combined with exercise training (Combined), on glucose (A) and insulin responses (B) to an oral glucose tolerance test in lean Zucker rats. Values are means ± SE for 8-12 animals/group. SE values (not shown) ranged from 3.4 to 8.3% of the mean for the glucose determinations (A) and from 5.5 to 20.5% of the mean for the insulin determinations (B). P < 0.05 vs. a sedentary group and b trandolapril group.

View larger version (23K): [in this window] [in a new window]   Fig. 2.   Effects of chronic treatment with trandolapril (Trand), exercise training (Exer), or trandolapril combined with exercise training (Combo) on the incremental areas under the curves (AUC) for glucose (A) and insulin (B) during an oral glucose tolerance test and the glucose-insulin index (C) in lean Zucker rats. Sed, sedentary control group. Data for the AUCs were taken from Fig. 1. The glucose-insulin index is calculated as the product of the glucose AUC and the insulin AUC for each animal (7). Values are means ± SE for 8-12 animals/group. P < 0.05 vs. a sedentary group and b trandolapril group.

Muscle glucose transport. To determine the cellular locus for this enhancement of peripheral insulin action, insulin-mediated glucose transport activity was assessed in isolated epitrochlearis and soleus muscles (Fig. 3). Rates of 2-DG uptake in the absence of insulin were not significantly different among the sedentary control, trandolapril only, exercise training only, and combined exercise training and trandolapril groups in the epitrochlearis (125 ± 13, 112 ± 8, 133 ± 21, and 130 ± 12 pmol · mg muscle¹ · 20 min¹, respectively) or in the soleus (350 ± 22, 335 ± 26, 369 ± 13, and 341 ± 22 pmol · mg muscle¹ · 20 min¹, respectively). In the epitrochlearis, exercise training alone induced a 33% increase in insulin-mediated 2-DG uptake (increase over basal) (Fig. 3A), and a 50% enhancement in this parameter was realized in the combined exercise training and trandolapril group. Trandolapril alone did not alter insulin-mediated 2-DG uptake in the epitrochlearis muscle of the lean Zucker rat.

View larger version (24K): [in this window] [in a new window]   Fig. 3.   Effect of chronic treatment with trandolapril, exercise training, or trandolapril combined with exercise training on insulin-mediated skeletal muscle glucose transport activity in lean Zucker rats. The net increase above basal for 2-deoxyglucose uptake due to insulin was assessed in the isolated epitrochlearis (A) and soleus muscle (B) preparations. Values are means ± SE for 8-12 animals/group. P < 0.05 vs. a sedentary group and b trandolapril group.

GLUT-4 protein and enzyme responses. In the epitrochlearis, trandolapril treatment alone did not alter whole homogenate GLUT-4 protein level (Fig. 4A). In contrast, exercise training alone induced a 66% increase, and the combination of exercise training and trandolapril treatment resulted in a 77% increase in the GLUT-4 protein level. In the soleus muscle, trandolapril alone was without effect, whereas exercise training alone or in combination with ACE inhibition caused 25% increases in GLUT-4 protein (Fig. 4B). Similarly, in the plantaris muscle, exercise training alone or in combination with trandolapril brought about 55% increases in GLUT-4 protein (Fig. 4C). These interventions did not induce significant alterations in GLUT-4 protein levels in the heart (data not shown).

View larger version (24K): [in this window] [in a new window]   Fig. 4.   Effect of chronic treatment with trandolapril, exercise training, or trandolapril combined with exercise training on whole muscle levels of GLUT-4 protein in lean Zucker rats. A: epitrochlearis; B: soleus; C: plantaris. Values are means ± SE for 8-12 animals/group. P < 0.05 vs. a sedentary group and b trandolapril group.

	DISCUSSION

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We have demonstrated in the present study that long-term endurance exercise training by lean Zucker rats, as is the case for other strains of rodents with normal insulin sensitivity (see review in Ref. 18), leads to significant increases in insulin-stimulated glucose transport activity in skeletal muscle that are associated with an increased level of the GLUT-4 glucose transport isoform and increased activities of enzymes involved in glucose phosphorylation (hexokinase) and glucose oxidation (citrate synthase). These findings provide further support for the concept that the expression of these factors involved in the uptake and metabolism of glucose by skeletal muscle are coregulated under the influence of enhanced neuromuscular activity (14, 22). More importantly, we have found that, in stark contrast to the response of insulin-resistant obese Zucker rats (26), 6 wk of treatment of insulin-sensitive lean Zucker rats with the ACE inhibitor trandolapril does not alter insulin action on skeletal muscle or the level of GLUT-4 protein in this tissue by itself nor does ACE inhibition alter the metabolic responses of these lean animals to endurance exercise training.

Our laboratory has recently demonstrated, in the insulin-resistant obese Zucker rat, that long-term ACE inhibition with the use of the long-acting agent trandolapril leads to significant increases in insulin action on skeletal muscle glucose transport activity that are associated with parallel increases in muscle levels of GLUT-4 protein and total hexokinase activities (19, 26). Furthermore, the beneficial effects of trandolapril treatment and endurance exercise training on muscle insulin action and GLUT-4 levels were completely additive in this rodent model of insulin resistance (26). It should be noted that these results were obtained in immature female obese Zucker rats and may not necessarily apply to males or to other insulin-resistant or hypertensive rat strains of other ages.

Although the present results, which show that, in the insulin-sensitive lean Zucker rat, chronic ACE inhibition does not modulate skeletal muscle insulin action, either by itself or combined with exercise training, are largely negative, in the context of our laboratory's previous findings in the obese Zucker rat, they have important implications. Evidence to date indicates that insulin action in insulin-resistant skeletal muscle from either obese (15, 17, 19, 26) or aged (6) animals is improved by acute (1-6 h) or chronic (2-6 wk) administration of an ACE inhibitor, likely due to the influence of the increased bradykinin that results from this intervention (6, 15-17). It would be of great interest if this same intervention could modulate insulin action in normal muscle, as it would suggest a role of bradykinin in the normal regulation of skeletal muscle glucose transport. However, the results of the present study clearly indicate that long-term ACE inhibition does not modulate insulin action in normal skeletal muscle. Collectively, these observations strongly suggest that a defect in the kallekrein/kinin system, which is impacted by ACE inhibitors (29), may be associated with insulin resistance of skeletal muscle glucose transport. What this defect might be and how it might manifest itself in insulin-resistant skeletal muscle is the subject of future investigations.

Although the vast majority of animal model and clinical studies investigating the effects of ACE inhibition on insulin-stimulated glucose disposal have used hypertensive and/or insulin-resistant subjects, there are a limited number of studies that have addressed the effects of ACE inhibition on this variable in normal, insulin-sensitive subjects. Uehara et al. (27) reported that, in normal dogs and humans, the acute administration of the short-acting ACE inhibitor captopril significantly increased whole body insulin-mediated glucose disposal. Moreover, two other groups (1, 9) showed that chronic administration of an ACE inhibitor (enalapril and fosinopril, respectively) also enhanced insulin-stimulated glucose disposal in normal human subjects. It should be emphasized, however, that these investigations quantified glucose disposal by using the euglycemic, hyperinsulinemic clamp, which can be influenced by hemodynamic alterations, such as occur with ACE inhibitors (29). It is likely that, in these normal subjects, the increased glucose disposal induced by ACE inhibition was hemodynamically mediated. In the present study, we used isolated muscle preparations from lean Zucker rats to quantify skeletal muscle glucose transport activity, and this in vitro assessment is, therefore, not under hemodynamic regulation. Therefore, in normal subjects, ACE inhibitors can influence in vivo glucose disposal via a hemodynamic mechanism but do not appear to have a direct effect on the glucose transport system in skeletal muscle.

Whereas the exercise training-induced enhancement of citrate synthase activity in the epitrochlearis, soleus, and plantaris muscles in the lean Zucker rat was not modified by concomitant ACE inhibition, in the latter two muscles (with a trend in the epitrochlearis), there was a significant interaction between exercise training and trandolapril treatment for increasing total hexokinase activity (Table 4). This is similar to our laboratory's previous observation in skeletal muscle from the obese Zucker rat (26). What underlies this particular interaction in either insulin-sensitive or insulin-resistant skeletal muscle that leads to a further enhancement of hexokinase activity is unclear. However, in the context of our observation that the combination of exercise training and trandolapril did not further increase insulin-stimulated 2-DG uptake or GLUT-4 protein in the soleus muscle of the lean Zucker rat, one could make the interpretation that an increase in hexokinase activity alone, without a concomitant upregulation of GLUT-4 protein expression, is not sufficient for an enhanced rate of insulin-stimulated glucose transport activity.

In conclusion, we have shown that, in contrast to insulin-resistant obese Zucker rats (19, 26), chronic ACE inhibition with trandolapril did not beneficially modify any of these parameters in the insulin-sensitive lean Zucker rat. The combination of exercise training and ACE inhibition in the lean Zucker rat, unlike in the obese Zucker rat (26), did not enhance insulin-stimulated muscle glucose transport activity or GLUT-4 protein beyond the levels attained with exercise training alone. Collectively, these results indicate that the positive interaction between endurance exercise training and ACE inhibition for enhancing skeletal muscle insulin action and GLUT-4 protein levels is restricted to conditions of insulin resistance and is not seen in insulin-sensitive muscle.