Introduction

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Chronic oral administration of the thiazolidinedione (TZD) troglitazone (chemical structure in Fujiwara et al., 1988) has been shown to markedly improve insulin sensitivity and glucose homeostasis in type 2 diabetic patients (Nolan et al., 1994; Kaneko, 1997; Schwartz et al., 1998) as well as in various animal models of diabetes and obesity (Fujiwara et al., 1988; Lee et al., 1994; Khoursheed et al., 1995). Troglitazone rapidly became an alternative drug for antidiabetic treatment in Japan and the U.S., but despite successful and extensive clinical use (Watkins and Whitcomb, 1998), the precise mechanisms via which troglitazone exerts its actions on glucose metabolism are not fully understood.

So far, it is known that TZD are potent agonists of the nuclear peroxisome proliferator-activated receptor- (PPAR), the activation of which modulates gene expression rates, resulting in adipocyte differentiation (Schoonjans et al., 1996; Spiegelman, 1998). Although good evidence is available for an essential role of PPAR in TZD-induced improvement of metabolic parameters (Lehmann et al., 1995; Berger et al., 1996; Spiegelman, 1998), it has not yet been established whether all TZD actions are mediated via PPAR-dependent modulation of gene transcription. Indeed, evidence accumulates that troglitazone can trigger metabolic responses within a short time range, which rather hints at the existence of transcription-independent TZD effects (Fujiwara et al., 1988; Lee and Olefsky, 1995; Bähr et al., 1996; Fulgencio et al., 1996; Fürnsinn et al., 1997a; Okuno et al., 1997; Park et al., 1998; Raman et al., 1998).

In rats, acute and therefore potentially transcription-independent troglitazone effects include a decrease in circulating glucose concentration (Fujiwara et al., 1988) and an increase in whole body glucose uptake within 30 min as measured by euglycemic-hyperinsulinemic clamp tests (Lee and Olefsky, 1995). Furthermore, studies on short-term troglitazone exposure in vitro revealed inhibition of gluconeogenesis in isolated hepatocytes (Fulgencio et al., 1996; Raman et al., 1998) as well as increased glucose uptake into cultured muscle cells and freshly prepared muscle specimens (Fürnsinn et al., 1997a; Park et al., 1998). Many short-term actions of troglitazone proved to be independent of concomitant insulin stimulation leading some authors to hypothesize an acute insulin-mimetic potential of the drug (Bähr et al., 1996; Park et al., 1998; Raman et al., 1998). At variance to that interpretation, we found in isolated rat soleus muscle that acute troglitazone exposure fails to stimulate intracellular glucose handling in an anabolic manner and, hence, lacks an acute insulin-like or insulin-sensitizing effect (Fürnsinn et al., 1997a). Our previous results suggest that the acute troglitazone-induced increase in glucose transport is associated with noninsulin-like catabolic stimulation of intracellular glucose fluxes as characterized by distinctly increased glycolytic flux and by glycogen depletion (Fürnsinn et al., 1997a).

With regard to lipid metabolism, acute troglitazone inhibition of long-chain fatty acid oxidation has been demonstrated in isolated rat hepatocytes and has been hypothesized to contribute to the antidiabetic efficacy of the drug by shifting fuel selection from lipid to carbohydrate (Fulgencio et al., 1996). Furthermore, troglitazone has been shown to block cholesterol biosynthesis in various cell types in vitro, obviously via a rapid and PPAR-independent mechanism (Wang et al., 1999). Most available studies on acute troglitazone action, however, focus on glucose metabolism (Fujiwara et al., 1988; Lee and Olefsky, 1995; Bähr et al., 1996; Fürnsinn et al., 1997a; Okuno et al., 1997; Park et al., 1998; Raman et al., 1998), thus, not accounting for the potential role of lipid metabolism. This study was undertaken to examine in parallel the direct effects of troglitazone on the utilization of palmitate and glucose in skeletal muscle, which is the quantitatively most important target tissue of insulin-stimulated glucose utilization (Baron et al., 1988).

	Materials and Methods

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Rats. Male Sprague-Dawley rats were purchased from the breeding facilities of the University of Vienna (Himberg, Austria). They were kept at an artificial 12-h light/dark cycle at constant room temperature. Conventional laboratory diet and tap water were provided ad libitum until the evening before sacrifice, when only food was withdrawn. Rats weighing approximately 140 g were sacrificed by cervical dislocation between 8:30 and 9:30 AM. All experiments were performed according to local law and to the principles of good laboratory animal care.

Long-Term Muscle Incubation (25-h). Immediately after sacrifice, two longitudinal soleus muscle strips per leg (i.e., four strips per rat) were prepared, weighed (approximately 25 mg/strip), and tied under tension on stainless steel clips as described previously (Crettaz et al., 1980). According to procedures used earlier (Stace et al., 1990; Fürnsinn et al., 1997b), muscles were immediately put into 50-ml Erlenmeyer flasks coated with BlueSlick solution (Serva, Heidelberg, Germany) and placed into a shaking water bath (six strips per flask; 37°C; 130 cycles/min). Each flask contained 20 ml of Cell Culture Medium 199 (5.5 mmol/l glucose; pH 7.35; cat. no. M-4530; Sigma, St. Louis, MO) with additions of 0.3% (w/v) fatty acid-free BSA, 5 mmol/l HEPES, 25,000 U/l penicillin G, and 25 mg/l streptomycin. Palmitate as dissolved in ethanol and troglitazone (provided by Sankyo, Tokyo, Japan) as dissolved in dimethyl sulfoxide (DMSO; Sigma) were added to give final concentrations of 300 µmol/l palmitate, 0.5% (v/v) ethanol, 0.1% (v/v) DMSO, and 0, 0.31, 0.63, 1.25, 2.5, or 5 µmol/l troglitazone. The effects of organic solvents were evaluated in the absence of palmitate in a separate experiment. An atmosphere of 95% O₂, 5% CO₂ was continuously provided within the flasks.

Short-Term Muscle Incubation (90-min). Muscles were put into coated 25-ml Erlenmeyer flasks that were placed into the water bath immediately after preparation (one strip per flask). Each flask contained a continuous atmosphere of 95% O₂, 5% CO₂ and 3 ml of Krebs-Ringer buffer solution (pH 7.35) with additions of 5.5 mmol/l glucose, 0.3% (w/v) BSA, 300 µmol/l palmitate, 0.8% (v/v) ethanol, 0.4% (v/v) DMSO, and 0, 5, 10, 20, 40, 80, 160, or 320 µmol/l troglitazone. The effects of organic solvents were evaluated in the absence of palmitate in a separate experiment.

Analytical Procedures. Net uptake of 2-deoxy-D-[2,6-³H]glucose, a glucose analog that is taken up by the cell where it accumulates as 2-deoxy-D-[2,6-³H]glucose-6 phosphate, was determined using [¹⁴C]sucrose as a marker of extracellular space by methods described previously (Fürnsinn et al., 1995). Under the applied experimental conditions, insulin-stimulated [³H]2-deoxyglucose uptake does not reach saturation within the incubation period of 60 min (data not shown). The net rate of glucose incorporation into glycogen is referred to as glycogen synthesis and was determined by measuring the conversion of [¹⁴C]glucose to [¹⁴C]glycogen as described previously (Crettaz et al., 1980). Rates of CO₂ production were calculated from the conversion of [¹⁴C]glucose or [¹⁴C]palmitate into ¹⁴CO₂, which was trapped with a solution containing methanol and phenethylamine (1:1) (Fürnsinn et al., 1995). Rates of lactate release were calculated from lactate accumulated in the incubation medium during the experiment. Medium lactate concentration was determined enzymatically, using the lactate dehydrogenase method (Engel and Jones, 1978). For the determination of muscle glycogen content prevailing at the end of the experiment, glycogen in the muscle lysate was completely degraded to glucose with amyloglucosidase (Dimitriadis et al., 1988). Glucose was then measured enzymatically by a commercial kit (Human, Taunusstein, Germany).

Statistics. All results are given as means ± S.E. and P values were calculated by two-tailed paired or unpaired Student's t test as appropriate. A P < .05 was considered significant.

	Results

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Effects of Organic Solvents. Exposure (25-h) to 0.5% ethanol and 0.1% DMSO did not significantly affect insulin-stimulated glucose metabolism as compared with control media without organic solvents (Table 1). In short-term experiments, short-term exposure to 0.8% ethanol and 0.4% DMSO shifted baseline glucose flux somewhat toward catabolic pathways (i.e., from glycogenesis to glycolysis; Table 1). The described effects of troglitazone can nevertheless be attributed to specific action of the drug, because concentrations of solvents were the same in all media including troglitazone-free controls.

Long-Term Troglitazone Exposure (5 µmol/l). The effects of long-term (25-h) exposure to 5 µmol/l troglitazone in M199 with 5.5 mmol/l glucose on parameters of fuel handling by rat soleus muscle are depicted in Fig. 1. Troglitazone markedly reduced palmitate oxidation to CO₂ to 31 ± 3 and 27 ± 3% of control under basal and insulin-stimulated (100 nmol/l) conditions, respectively (Fig. 1A). In parallel, glucose oxidation was significantly inhibited to 52 ± 5 and 51 ± 4%, respectively (Fig. 1B). Hence, the relative decrease in oxidation to CO₂ was significantly more pronounced for palmitate than for glucose under both basal and insulin-stimulated conditions (P < .005 each).

View larger version (43K): [in this window] [in a new window]   Fig. 1.   Fuel metabolism in soleus muscle strips from healthy Sprague-Dawley rats exposed to 0 (Control) or 5 µmol/l troglitazone for 25 h. Rates of basal and insulin-stimulated (100 nmol/l) CO2 production from palmitate (A) and glucose (B), lactate release (C), [3H]2-deoxyglucose transport (D), and glycogen synthesis (E), as well as glycogen content as determined after the experiment (F) are depicted. Means ± S.E.; n = 10-12 each; a, P < .02; b, P < .001; c, P < .0001 by paired Student's t test.

View larger version (43K): [in this window] [in a new window]   Fig. 2.   Fuel metabolism in soleus muscle strips from healthy Sprague-Dawley rats exposed to different concentrations of troglitazone for 25 h. Rates of insulin-stimulated (100 nmol/l) CO2 production from palmitate (A) and glucose (B), lactate release (C), [3H]2-deoxyglucose transport (D), and glycogen synthesis (E) as well as glycogen content as determined after the experiment (F) are depicted as differences versus an intraindividual control incubated without troglitazone (control values listed in Table 2). Means ± S.E.; n = 6-24 each; a, P < .05; b, P < .01; c, P < .005; d, P < .001; e, P < .0001 by paired Student's t test.

Long-Term Troglitazone Exposure, Dose-Response-Curve. Figure 2 depicts a dose-response curve for long-term muscle incubation with troglitazone. Because the experiment described above clearly indicated that troglitazone action was independent of concomitant insulin stimulation in vitro, dose dependence was tested under insulin-stimulated conditions only. The data are given as differences versus an intraindividual control value as determined in the absence of troglitazone in the same soleus muscle incubation assay (control values depicted in Table 2).

Short-Term Troglitazone Exposure. The dose-dependent effects of short-term (90-min) troglitazone exposure on various parameters of soleus muscle fuel handling (Fig. 3) are also given as differences versus an intraindividual control value as depicted in Table 2.

View larger version (37K): [in this window] [in a new window]   Fig. 3.   Fuel metabolism in soleus muscle strips from healthy Sprague-Dawley rats exposed to different concentrations of troglitazone for 90 min. Rates of basal and insulin-stimulated (30 nmol/l) CO2 production from palmitate (A) and glucose (B), lactate release (C), [3H]2-deoxyglucose transport (D), and glycogen synthesis (E), as well as glycogen content as determined after the experiment (F) are depicted as differences versus an intraindividual control incubated without troglitazone (control values listed in Table 2). Means ± S.E.; n = 5-17 each; a, P < .05; b, P < .01; c, P < .005; d, P < .001; e, P < .0001 by paired Student's t test.

	Discussion

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Our previous study in isolated rat soleus muscle (Fürnsinn et al., 1997a) showed that 90-min troglitazone exposure stimulates glucose metabolism in a noninsulin-like catabolic manner as characterized by marked stimulation of anaerobic glycolysis in association with increased glucose transport but glycogen depletion. This study sheds more light on the direct interaction of troglitazone with skeletal muscle, demonstrating a distinct potential of the drug to directly inhibit CO₂ production from glucose as well as from palmitate. Blunted oxidative fuel utilization is reflected by a marked increase in lactate release, indicating that under troglitazone exposure, the energy demands of isolated muscle are to an increased extent covered via anaerobic glycolysis. Such shift toward anaerobic glucose metabolism necessarily enhances the requirement for glycolytic substrate, which is apparently covered by an increment in the rate of glucose uptake and by reduced glucose flux into glycogen stores.

At present, troglitazone is believed to exert its therapeutic action via PPAR-dependent modulation of gene transcription, finally resulting in insulin sensitization and improved glucose homeostasis (Lehmann et al., 1995; Berger et al., 1996; Spiegelman, 1998). Insulin sensitization, however, can hardly account for the effects on fuel metabolism described in this study, because troglitazone potently acted in the absence of insulin. Furthermore, the distinct and relatively rapid effect on palmitate oxidation to CO₂ (to 64 and 48% within 90 min under basal and insulin-stimulated conditions, respectively) raises the possibility of a transcription-independent mechanism of action. It can, therefore, not be excluded that an as yet unknown biochemical mechanism underlies acute actions of troglitazone in vivo (Fujiwara et al., 1988; Lee and Olefsky, 1995) as well as its direct actions on isolated muscle, with the dramatic reduction of CO₂ production hinting at the inhibition of a common step of mitochondrial glucose and palmitate oxidation. In line with that hypothesis, evidence from isolated rat hepatocytes indicates that a troglitazone-induced decrease in long-chain fatty acid oxidation occurs in association with a highly oxidized mitochondrial redox state (Fulgencio et al., 1996). Inhibition of the enzyme long-chain acyl-coenzyme A synthetase by the troglitazone metabolite M1 as found in the hepatocyte model (Fulgencio et al., 1996) can be excluded with regard to isolated skeletal muscle because evidence suggests that troglitazone metabolism to M1 occurs in liver rather than muscle (Kawai et al., 1997). Furthermore, the inhibition of long-chain acyl-coenzyme A synthetase should specifically blunt CO₂ production from palmitate but not from glucose. Nevertheless, it should be noted that in our experimental setting, CO₂ production from palmitate was more markedly reduced than from glucose, indicating a troglitazone-induced increase in the ratio of glucose/palmitate converted into CO₂. Despite a reduction in absolute rates of conversion to CO₂ of both glucose and palmitate, our results, therefore, demonstrate a relative shift in fuel utilization from lipid to carbohydrate.

It is of note that both a shift in fuel utilization from lipid to carbohydrate and the induction of a more oxidized mitochondrial state are attributes of other antidiabetic compounds structurally unrelated to troglitazone (Wolf, 1992; Roy et al., 1997; Deems et al., 1998). In spite of such similarities between the actions of troglitazone and other antidiabetic agents, these findings do not allow for final conclusions on the potential contribution of acute troglitazone effects observed in vitro to its antidiabetic efficacy with chronic administration in vivo. The troglitazone concentration required to trigger a significant response in 25-h exposed muscle is below that circulating in plasma from successfully treated diabetic rodents (approximately 0.6 µmol/l; Lee et al., 1994). The increase in troglitazone efficacy with prolonged exposure time (25 h versus 90 min) also underlines the possibility that direct troglitazone effects on muscle fuel oxidation may be of relevance in patients subject to chronic oral treatment. All assumptions on effective troglitazone concentrations in vitro and in vivo are, however, hampered by its unknown biological availability. One of many factors that putatively affect troglitazone availability is its binding to proteins (Sibukawa et al., 1995), and different protein concentrations used in vitro can explain the variations in effective drug concentrations observed in this versus other studies (Fulgencio et al., 1996; Fürnsinn et al., 1997a; Okuno et al., 1997). Additional factors that may affect troglitazone action include facilitation of drug availability by organic solvents in vitro (DMSO, ethanol) or by lipoprotein particles in vivo.

It is of note that results from muscle specimens exposed to troglitazone concentrations above 80 µmol/l for 90 min suggest the operation of another mechanism of troglitazone action that obviously overcomes the inhibitory effect of the drug on fuel oxidation prevailing at lower concentrations. The response to high troglitazone concentrations can be interpreted as distinct catabolic action on muscle fuel metabolism, because increased CO₂ production from palmitate is associated with marked stimulation of both aerobic and anaerobic glycolysis as well as with glycogen depletion. Although a role for such catabolic stimulation in the therapeutic action of troglitazone in vivo can not be excluded, an unspecific response due to high ambient drug concentrations in vitro seems rather conceivable. In line with such interpretation, the relative decrease in insulin-stimulated palmitate oxidation to CO₂ observed between 160 and 320 µmol/l troglitazone may indicate advanced toxic damage.

Taken together, the study describes acute inhibition by troglitazone of oxidative fuel metabolism in isolated rat soleus muscle, and therefore demonstrates direct troglitazone actions on the quantitatively most important target tissue of insulin. These previously unknown effects of troglitazone are rapid in occurrence and independent of concomitant insulin stimulation. Their contribution to the antidiabetic efficacy of compound in vivo as well as the underlying mechanism of action remain to be elucidated.