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Agonist Tesaglitazar
Diabetes and Obesity Research Program (B.D.H., S.M.F., E.W.K., G.J.C.), Garvan Institute of Medical Research, Sydney, New South Wales 2010, Australia; and AstraZeneca R&D (N.D.O.), S-431 83 Mölndal, Sweden
Address all correspondence and requests for reprints to: Stuart Furler, Ph.D., The Garvan Institute of Medical Research, St. Vincent’s Hospital, 384 Victoria Street, Sydney, New South Wales 2010, Australia. E-mail: s.furler{at}garvan.org.au.
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Abstract |
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agonist tesaglitazar on whole-body insulin sensitivity and NEFA clearance into epididymal white adipose tissue (WAT), red gastrocnemius muscle, and liver in rats with dietary-induced insulin resistance. Wistar rats were fed a high-fat diet (59% of calories as fat) for 3 wk with or without treatment with tesaglitazar (1 µmol·kg–1·d–1, 7 d). NEFA clearance was measured using the partially metabolizable NEFA tracer, 3H-R-bromopalmitate, administered under conditions of basal or elevated NEFA availability. Tesaglitazar improved the insulin sensitivity of high-fat-fed rats, indicated by an increase in the glucose infusion rate during hyperinsulinemic-euglycemic clamp (P < 0.01). This improvement in insulin action was associated with decreased diglyceride (P < 0.05) and long chain acyl coenzyme A (P < 0.05) in skeletal muscle. NEFA clearance into WAT of high-fat-fed rats was increased 52% by tesaglitazar under basal conditions (P < 0.001). In addition the PPAR/ agonist moderately increased hepatic and muscle NEFA utilization and reduced hepatic triglyceride accumulation (P < 0.05). This study shows that tesaglitazar is an effective insulin-sensitizing agent in a mild dietary model of insulin resistance. Furthermore, we provide the first direct in vivo evidence that an agonist of both PPAR and PPAR increases the ability of WAT, liver, and skeletal muscle to use fatty acids in association with its beneficial effects on insulin action in this model. |
Introduction |
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In recent years, agonists of the peroxisome proliferator-activated receptors (PPARs), of the and subtypes (PPAR and PPAR), have been found to have beneficial effects on insulin action. Indeed, the insulin-sensitizing effects of PPAR agonists have been demonstrated in a wide variety of experimental models, and they are now used clinically for the treatment of type 2 diabetes (1). Agonists of PPAR are best known as antihyperlipidemic agents but have also recently been shown to improve insulin sensitivity in animal models (2, 3) as well as in man (4, 5, 6, 7).
PPARs are transcription factors that have important effects on lipid homeostasis via regulation of the expression of genes involved in lipid metabolism. PPAR is predominantly expressed in the liver and regulates the transcription of genes involved in hepatic fatty acid uptake and oxidation, including CD36/FAT, acyl coenzyme A (CoA) synthetase, acyl-CoA oxidase, and carnitine palmitoyl transferase I (8). PPAR is expressed mainly in white adipose tissue (WAT), where its activation stimulates the expression of genes involved in fatty acid uptake and storage, including lipoprotein lipase (LPL), CD36/FAT, fatty acid-binding protein aP2, ACS, and phosphoenolpyruvate carboxykinase (8). Despite relatively low mRNA expression, there are also significant levels of PPAR and PPAR protein in skeletal muscle (9).
It is generally believed that the ability of PPAR agonists to up-regulate lipid metabolism in WAT and liver is central to their insulin-sensitizing effects (10). Thus, the activation of PPARs is thought to cause sequestration of lipid into WAT, for storage, or into the liver for oxidation. These actions putatively reduce the amount of lipid available to skeletal muscle, where it may otherwise accumulate and interfere with insulin action (2, 3, 11, 12, 13).
The above views have become widely accepted despite a paucity of direct in vivo evidence demonstrating that PPAR agonists do, in fact, alter lipid fluxes to WAT and/or liver. A previous study (14) demonstrated that treatment with a PPAR agonist enhances the ability of adipose tissue to store nonesterified fatty acid (NEFA) in anesthetized obese Zucker rats. In the present study, we have investigated, in conscious animals, the capacity of tesaglitazar, a novel PPAR/ agonist (15), to modulate both total NEFA uptake and storage in WAT, liver, and muscle in the high-fat-fed rat (a nongenetic model of insulin resistance). The selectivity and potency of tesaglitazar, compared with specific PPAR and PPAR agonists, has been reported previously (16). To determine total tissue NEFA uptake, we employed the partially metabolizable fatty acid tracer, [9,10-3H]-(R)-2-bromopalmitate (3H-R-BrP) (17). We have recently used this methodology to show that the skeletal muscle of high-fat-fed insulin-resistant rats has an enhanced ability to take up fatty acids, which may contribute to accumulation of intramyocellular lipid and associated detrimental effects on insulin action (18). In that study, we observed different metabolic responses in liver and WAT when systemic NEFA levels were elevated. Consequently, we also investigated PPAR agonist action when plasma NEFA concentrations were normal and elevated.
The specific aims of the current study were: 1) to assess the ability of tesaglitazar to improve insulin action in a mild dietary model of insulin resistance; 2) to determine whether activation of both PPAR and PPAR regulates NEFA utilization in WAT such that lipid is sequestered away from muscle; and 3) to investigate the possibility that PPAR and PPAR activation alters the ability of skeletal muscle to dispose of NEFA.
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Materials and Methods |
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Experiments were performed on male Wistar rats purchased from Animal Resources Centre, Perth, Western Australia. Rats were housed in a temperature-controlled (22 ± 1 C) environment with a 12-h light, 12-h dark cycle (lights on at 0600 h). After a 1-wk acclimatization period, rats were fed a high-fat diet (59% fat, 21% protein, and 20% carbohydrate, expressed as a percent of total dietary calories) for 3.5 wk to induce insulin resistance.
Surgery
Anesthesia was induced with 5% and maintained with 1–2% halothane in oxygen. Chronically indwelling cannulae were inserted into a jugular vein and carotid artery as previously described (19). Suture lines were infiltrated with bupivicane (0.5 mg/100 g). Buprenorphine (0.003 mg/100 g) was administered sc postoperatively. Rats recovered for 9–10 d before the acute study.
Drug administration
In the 7 d before the study, one group of rats (HF-PPAR) was gavaged with tesaglitazar, 1 µmol·kg–1·d–1, suspended in vehicle consisting of 0.5% (wt/vol) methylcellulose in water. A second group (HF) was gavaged with vehicle alone. Animals were dosed at 0930–1030 h each morning in a gavage vol of 2.5 ml·kg–1. Previous studies in ob/ob mice (16) had indicated that this dosage regimen (1 µmol·kg–1·d–1 for 1 wk) normalized both the hyperglycemia and hypertriglyceridemia usually associated with ob/ob mice, and increased the whole-body insulin sensitivity of obese Zuckers to that of lean controls, as assessed by clamp studies.
Treatment before tracer administration
The effects of tesaglitazar on tissue-specific NEFA clearance and metabolism were measured under varying conditions of substrate supply, by infusing 3H-R-BrP and 14C-palmitate (14C-P) tracers under either postabsorptive (basal) conditions or conditions where NEFA availability was elevated by combined infusion of intralipid and heparin (intralipid+heparin). Intralipid (20%; Baxter Healthcare, Sydney, Australia) was mixed with heparin (71.5 U/ml, AstraZeneca, Sydney, Australia) and infused at a constant rate of 1.3 ml/h (294 µmol triglyceride/h) via the jugular cannula. For the intralipid+heparin groups, tracer administration started 30 min after the commencement of the intralipid+heparin infusion. A subset of rats underwent hyperinsulinemic-euglycemic clamp to measure the effect of tesaglitazar on insulin sensitivity. These rats were infused with insulin (Actrapid, Novo Nordisk, Copenhagen, Denmark) via the jugular cannula at a constant rate of 0.25 U·kg–1·h–1 together with a variable rate of 30% glucose to maintain euglycemia (20).
Tracer administration
Rats were administered a mixture of 3H-R-BrP and [U-14C]-palmitate (14 , DuPont, Boston, MA). The 3H-R-BrP tracer was produced at AstraZeneca, Mölndal, Sweden. The synthesis of racemic [9,10-3H]-2-bromopalmitic acid and subsequent resolution of the R-isomer have been previously described (17). Each rat was infused with approximately 25 x 106 dpm 3H-R-BrP and 13 x 106 dpm 14C-P in 1 ml vehicle. Tracers were delivered in saline, complexed with BSA (17), and infused at a constant rate into the jugular cannula of conscious rats over a 4-min period.
Plasma samples
Arterial blood samples (
400 µl) were collected at baseline and directly before tracer administration. Smaller samples (200 µl) were also taken at 5-min intervals during infusion of intralipid+heparin and at 1, 2, 3, 4, 5, 6, 8, 12, and 16 min after the start of tracer infusion. All blood samples were immediately centrifuged, and the separated plasma was frozen by immersion in liquid nitrogen and stored at –20 C until analyzed. Erythrocytes from the samples taken before tracer administration were resuspended in saline and returned to the animal to minimize blood loss. Throughout the experiment, the arterial cannula was kept patent by a slow constant infusion (0.6 ml/h) of 20 mM sodium citrate in saline.
Tissue samples
After collection of the final blood sample, rats were killed with an overdose (60 mg) of pentobarbitone injected via the carotid cannula. Samples of red gastrocnemius muscle (RG) and red quadriceps muscle, liver, and epididymal WAT were rapidly dissected, freeze-clamped with aluminum tongs precooled in liquid nitrogen, and stored at –70°C awaiting analysis.
Plasma tracer concentrations
The method for isolation of 3H-R-BrP and 14C-P from total 3H and 14C plasma activities has been described previously (17). Briefly, an initial acid lipid extraction, using a mixture of isopropanol-hexane-0.5 M H2SO4 (40:10:1) was followed by a polarity separation step, under alkaline conditions. The latter procedure predominantly partitioned esterified fatty acids into a hexane phase, and NEFAs in anionic form (including the 3H-R-BrP and 14C-P tracers) into an alcohol phase. Small corrections (<10%), based on separation of NEFA and esterified fatty acid standards, were applied for incomplete partitioning of tracer.
Tissue tracer content
Tissue samples were homogenized in chloroform:methanol (2:1) using a glass hand-held homogenizer. An aliquot of this homogenate was taken to determine the total activity of the 3H label. The remaining tissue homogenate was spun at 3500 x g for 15 min. The resultant supernatant was separated into aqueous and organic phases by the addition of 1 ml distilled water and an additional 10-min spin at 3500 x g. The 14C activity in the organic phase was used to measure the incorporation of tracer into intracellular lipid pools.
Activities of 3H and 14C in appropriate plasma and tissue fractions were measured on a Beckman LS 6000SC liquid-scintillation counter (Beckman Instruments, Fullerton, CA) using a dual-label protocol.
Plasma lipids
Plasma NEFA concentration was determined using an acyl-CoA oxidase based colorimetric kit (WAKO FA-C; WAKO Pure Chemical Industries, Osaka, Japan). Plasma triglyceride (TG) content was measured using an enzymatic colorimetric technique (Peridochrom Triglycerides GPO-PAP, Roche Molecular Biochemicals, Mannheim, Germany).
Tissue lipid content
Hepatic TG and diglyceride (DAG) content and the TG content of RG from basal animals were determined using an HPLC method adapted from that of Homan et al. (21). The lipid extract of tissue samples was separated on a Sherisorb S5W silica column (Waters, Milford, MA) using a mobile phase combining three solvent mixtures: 1) heptane:tetrahydrofurane (99:1, vol/vol); 2) acetone:dichloromethane (2:1, vol/vol); and 3) 2-propanol: (7.5 mM acetic acid, 7.5 mM ethanolamine, in water) (85:15 vol/vol). TGs were detected using an evaporative light-scattering device (Polymer Laboratories PL-ELS 1000, Church Stretton, UK) and quantified by measurement of peak area and comparison to known standards using Chromeleon software (version 4.1, Dionex-Softron, Germering, Germany).
Lipids were extracted from samples of red quadriceps muscle and the DAG content determined using a radioenzymatic DAG assay kit (Amersham Pharmacia Biotech, Amersham, UK) as previously described (22).
The long chain acyl-CoA (LCACoA) content of liver and red quadriceps samples was measured using a fluorometric assay adapted from the method of Antinozzi et al. (23). Tissue samples (50 mg) were homogenized in 10% trichloroacetic acid and centrifuged at 10,000 x g for 10 min. The LCACoA-containing pellet was washed by resuspension and centrifugation (10,000 x g, 10 min) with diethyl ether and then water. CoA was hydrolyzed from fatty acids by resuspending the pellet in 10 mM dithiothreitol, raising the pH to 11.5 with KOH, and heating to 55 C for 10 min. Samples were buffered to a final concentration of 50 mM KH2PO4, neutralized by adding 1 M HCl, and the insoluble fatty acids removed by centrifugation (13,000 x g, 10 min). Aliquots of sample supernatant and CoA standards were added to reaction buffer containing 500 µM nicotinamide adenine dinucleotide and 100 µM -ketoglutarate. Fluorescence was read using a fluorescence spectrophotometer (F4010, Hitachi, Tokyo, Japan) at an excitation wavelength of 340 nm and an emission wavelength of 460 nm, before and 10–20 min after the addition of -ketoglutarate dehydrogenase (35 mU, Sigma Chemical Co, St. Louis, MO). LCACoA content was calculated using the increase in fluorescence and comparison to CoA standards (Sigma Chemical Co).
Calculations
Whole-body clearance of 14C-P (MCRp) and the tissue clearance rates of 3H-R-BrP and 14C-P (K*f and K'fs, respectively) were calculated from tissue content and arterial plasma profile of radiolabeled tracer and metabolic products as previously described (17, 18, 24). K*f is proportional to the clearance rate of circulating NEFA (17), but the constant of proportionality (LC*) is not equal to 1. The results presented here have been corrected using previously determined values for LC* (24). Kf*/LC* is an index of total tissue clearance. K'fs is an estimate of the component of tissue clearance that is directed to lipid storage products.
Because high-fat feeding of rats tends to increase the mass of both the liver and WAT, FA clearance into these tissues was expressed per whole-liver and adipose depot, respectively, to reflect their influence on whole-body uptake.
Statistical analysis
Statistical analysis was performed using a commercial software package (StatView; Abacus Concepts/Brainpower, Berkeley, CA). Comparisons of two groups were performed by Student’s t test. Comparison of three groups was performed by one-way ANOVA incorporating a Fisher’s protected least-significant-difference post hoc test. Group results are presented as means ± SEM. P < 0.05 was considered statistically significant.
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Results |
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). This beneficial effect of tesaglitazar on insulin sensitivity was associated with a marked reduction in basal plasma insulin levels (Table 1) to values similar to those previously reported for rats fed a conventional high-carbohydrate chow diet [29.9 ± 5.6 mU·liter–1 (18)]. The normoglycemic plasma glucose levels were unaffected (Table 1). Tesaglitazar treatment did not affect body weight (HF, 370 ± 5 vs. HF-PPAR, 369 ± 5.5 g) or epididymal WAT mass (HF, 4.1 ± 0.5 vs. HF-PPAR, 3.6 ± 0.3 g) but did increase liver weight (HF, 15.3 ± 0.8 vs. HF-PPAR, 18.0 ± 0.8 g, P < 0.05).
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). However, during the infusion of intralipid+heparin, high-fat-fed rats treated with tesaglitazar exhibited markedly lower plasma TG and NEFA concentrations, associated with increased whole-body NEFA clearance (Table 1).
Tissue-specific NEFA metabolism
The intrinsic ability of a tissue to take up a circulating substrate is described by the so-called tissue clearance. This parameter depends on factors such as substrate transporters in the plasma membrane and substrate sequestration due to intracellular enzyme activity, and not on the circulating level of substrate. NEFA clearance (Kf*) into WAT, liver, and RG were measured in this study using the accumulation of the partially metabolizable fatty acid tracer, 3H-R-BrP. Clearance of NEFA to intracellular storage (K'fs) was determined using tissue accumulation of 14C-P in the organic extract of the tissues (as described in Materials and Methods). As previously reported (18), K'fs is lower in red muscle (3.0 ± 0.3 ml·min–1·100 g–1) and liver (6.3 ± 0.6 ml·min–1) of rats fed a chow diet compared with the fat-fed animals considered here.
Under basal conditions, tesaglitazar-treated animals showed a markedly increased NEFA clearance in epididymal WAT compared with untreated animals (Fig. 2A). This was accompanied by a parallel increase in NEFA clearance into storage in WAT (Table 2).
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). In contrast, intralipid and heparin had no further effect on the increased NEFA clearance observed in WAT of the tesaglitazar-treated animals.
During infusion of intralipid+heparin, hepatic NEFA clearance was moderately elevated in the tesaglitazar-treated vs. control rats (Fig. 2B). This higher hepatic NEFA clearance was not associated with an elevated NEFA clearance into storage (Table 2), suggesting enhanced hepatic fatty acid oxidation.
In the RG, there was no effect of the PPAR / agonist on NEFA clearance under basal conditions, but a small increase was observed in the heparin+intralipid group. As in the liver, there was no parallel effect on NEFA clearance into storage in muscle under this condition (Fig. 2C and Table 2), again suggesting that increased total NEFA clearance was associated with an increase in NEFA oxidation.
Tissue lipid content
The current results confirm previous findings that the high-fat diet used in these studies raises the hepatic (Fig. 3, A–C) and muscular (Fig. 3, D–F) content of TGs, DAGs, and LCAC-CoA. Tesaglitazar treatment normalized DAG and total LCAC-CoA levels but did not significantly lower TG in RG (Fig. 3, D–F). In the liver, the accumulation of TG (Fig. 3A) and DAG (Fig. 3B) in high-fat-fed rats was reduced by tesaglitazar. In contrast to its effects in skeletal muscle, tesaglitazar increased the hepatic LCACoA content of high-fat-fed rats (Fig. 3C).
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Discussion |
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and PPAR (15) is an effective insulin-sensitizing agent in a nongenetic model of insulin resistance. We have also investigated some of the alterations in lipid metabolism that accompany the amelioration of insulin resistance by this PPAR agonist. In previous studies (11, 14), we have demonstrated that PPAR agonists enhance the ability of insulin to suppress NEFA mobilization. In the present study, we investigated whether PPAR agonists can also reduce lipid supply to some tissues by increasing clearance of NEFA in others. We have investigated tissue-specific effects of tesaglitizar under conditions of both low and high NEFA availability, because we had previously found that altered NEFA availability can modulate the effect of a high-fat diet on NEFA metabolism (18). Here, we used an infusion of heparin/TG emulsion to elevate circulating NEFA, but our results are applicable to other situations where NEFA levels are systemically elevated. These include physiological states, such as prolonged fasting and intense exercise, and pathophysiological states such as diabetes.
Tesaglitazar had a major effect to increase the NEFA clearance into WAT of high-fat-fed rats under basal conditions (Fig. 1A) and had similar effects on NEFA clearance into storage (Table 2). It is likely that this increase in NEFA clearance is the functional result of the well-described effects of PPAR agonists to stimulate the transcription of genes involved in NEFA transport and storage in WAT (25).
Interestingly, when untreated HF rats were infused with intralipid and heparin, NEFA clearance into WAT increased to levels similar to that seen in HF-PPAR rats under basal conditions. This effect may be due to the elevated insulin levels of the HF intralipid+heparin rats (Table 1). It has recently been shown that the fatty-acid transporter FAT/CD36 is present in an intracellular compartment from which it can be acutely translocated to the plasma membrane by insulin, to stimulate NEFA uptake in muscle (26). Similarly, the lower insulin levels in tesaglitazar-treated rats would tend to decrease NEFA uptake, possibly limiting the direct actions of the PPAR agonist. Although other explanations are possible, this may explain why there was no further effect of tesaglitazar on WAT NEFA clearance in intralipid+heparin infused rats (Fig. 2A).
Previously, Oakes et al. (14) showed that the greatly improved insulin sensitivity of obese Zucker rats after treatment with the PPAR agonist darglitazone was associated with a normalization of the markedly elevated (6-fold greater than lean controls) plasma TGs seen in obese Zucker rats. In the current study, tesaglitazar reduced circulating TGs despite the fact that high-fat-fed rats do not display hypertriglyceridemia (Table 1). Thus, in the grossly obese, severely insulin-resistant, and hyperlipidemic Zucker rats, the beneficial effects of PPAR agonists are particularly apparent because of the combination of the efficacy of the compound and the severity of the disorders. In the current model of mild insulin resistance, without hypertriglyceridemia and gross obesity, the effects of tesaglitazar are more modest but qualitatively similar to those observed in obese fa/fa Zucker rats (Oakes et al., unpublished observations). They include an increase in the ability of WAT to take up and store NEFA and a reduction in the systemic availability of fatty acids in the form of TG.
The fact that HF-PPAR-treated rats achieved lower plasma TG concentrations than HF rats when infused with the same amount of intralipid+heparin (Table 1) suggests that tesaglitazar increases TG clearance in addition to NEFA clearance. Increased TG clearance could result from increased hydrolysis, because PPAR agonists are known to increase the gene expression of LPL (27). In addition, both PPAR agonists (27) and PPAR agonists (28, 29) inhibit the expression of Apo-CIII, an apolipoprotein that inhibits the hydrolyzing action of LPL. Apo-CIII also inhibits the uptake of TG-rich lipoprotein remnants by the liver (30). Inhibition of Apo-CIII expression would therefore promote clearance of TG from the circulation both by increasing hydrolysis and increasing removal of TG-rich particles without prior lypolysis.
The liver was markedly increased in size by tesaglitazar treatment, which is a typical response to PPAR ligands in rodents (31). Despite this confirmation of PPAR activation, tesaglitazar treatment had relatively subtle effects on hepatic NEFA metabolism as measured by 3H-R-BrP and 14C-P tracers. Tesaglitazar-treated rats demonstrated a small increase in hepatic NEFA clearance compared with HF rats under conditions of elevated NEFA availability (Fig. 2B). However, in contrast to WAT, there was no corresponding effect of tesaglitazar on hepatic NEFA clearance into storage (Table 2). This suggests that the increase in NEFA clearance into the liver with tesaglitazar was accompanied by a similar increase in NEFA oxidation. This is consistent with the expected effect of PPAR activation, which up-regulates the transcription of genes involved in hepatic uptake, activation, and oxidation of fatty acids. Considering that in this study the liver was found to be responsible for approximately 50% of whole-body NEFA uptake, this effect of tesaglitazar would represent a significant effect on whole-body NEFA utilization over time.
A reduced capacity of insulin to suppress hepatic glucose output is a component of the whole-body insulin resistance observed in high-fat-fed rats (32). This aspect of hepatic insulin sensitivity is inversely correlated with liver TG content (33, 34). Tesaglitazar decreased hepatic TG and DAG content (Fig. 3), probably by reducing systemic lipid availability and/or by increasing hepatic NEFA oxidative capacity. However, liver LCACoA content of high-fat-fed rats was increased by tesaglitazar treatment (Fig. 3), a situation that has been previously associated with decreased hepatic insulin action (34, 35, 36, 37, 38). However, it should be noted that this measurement of total LCACoAs does not differentiate between LCACoA in the cytosol and those within organelles. We suggest that tesaglitazar-induced increase in hepatic LCACoA is a result of the established action of PPAR agonists to increase the number of peroxisomes in the rodent liver (31). The expanded peroxisome population would be expected to increase the liver’s total content of CoA species; but if they are confined within the peroxisomes, these lipid moieties might not be able to interfere with insulin signaling or carbohydrate metabolism.
Tesaglitazar did not significantly reduce the accumulation of TG in skeletal muscle of high-fat-fed rats, as has previously been observed with a 2-wk thiazolidinedione treatment (3), presumably due to the shorter (1 wk) treatment period used in the present studies. Muscle TG is often used as a marker of insulin resistance, but there has been no demonstration that TG per se interferes with insulin action. However, tesaglitazar treatment did reduce the elevated content of DAG and LCACoA in the skeletal muscle of high-fat-fed rats to levels similar to that of insulin sensitive rats fed a standard diet (Fig. 3). These lipid intermediates are important molecules that have been shown to interfere with various aspects of insulin signaling and carbohydrate metabolism and are likely to contribute to the detrimental effects of im lipid accumulation on insulin action (39, 40).
Recently, we have shown that insulin resistance induced by feeding rats a high-fat diet is associated with an increase in NEFA clearance into skeletal muscle, which is likely to contribute to the accumulation of lipid within this tissue (18). Given that PPARs are thought to work by reducing the lipid accumulation within muscle, we were therefore interested to see whether PPAR agonists might act to reverse this effect of high-fat feeding on muscle NEFA clearance. However, the data from the RG (Fig. 3) suggested that this was not the case. In fact, tesaglitazar treatment induced a moderate increase in NEFA clearance into this tissue when circulating lipid levels were elevated. As was the case in the liver, it was not accompanied by a parallel increase in NEFA clearance into storage, indicating that the increased NEFA clearance into muscle under this condition was directed to oxidation, a possible consequence of PPAR activation.
The interpretation of our tracer results in liver and muscle (regarding oxidation) is based on previous validation studies (41), in which rates of oxidation were directly manipulated by pharmacological ß-oxidation blockade (etomoxir administration) and by skeletal muscle contractions. The response pattern observed here (increased 3H-R-BrP clearance with no change in 14C-P clearance) is the same as that observed in muscle when NEFA oxidation was increased by contractions. This interpretation of our in vivo results is also supported by previous studies in cellular systems (28, 42).
The current study is predominantly an investigation of NEFA, not TG, metabolism. The uptake of fatty acids derived from the local hydrolysis of circulating TG by the action of LPL has not been assessed. Both PPAR and PPAR agonists modulate the expression of LPL at the level of mRNA (43), and it is likely therefore that tesaglitazar alters TG-derived fatty acid metabolism. Methodology for assessing this pathway in individual tissues in vivo is under development (44).
In conclusion, this study shows that the novel PPAR/ agonist, tesaglitazar, improved insulin sensitivity in a nongenetic model of mild insulin resistance, the high-fat-fed rat. Moreover we have significantly clarified the mode of action of tesaglitazar by investigating tissue-specific alterations in NEFA metabolism associated with its beneficial effects on insulin action. Tesaglitazar reduced plasma TG levels, hepatic TG accumulation, and DAG and LCACoA levels in skeletal muscle of high-fat-fed rats. This was probably via a reduction in fatty acid supply, because there was no reduction in the intrinsic ability of these tissues to take up and store NEFA. Most importantly, we have provided the first direct in vivo evidence that a PPAR/ agonist increases the ability of both WAT and liver to use fatty acids, thereby sparing muscle from the detrimental effects of lipid oversupply.
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Acknowledgments |
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Footnotes |
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Abbreviations: CoA, Coenzyme A; DAG, diglyceride; 3H-R-BrP, [9,10-3H]-(R)-2-bromopalmitate; LCACoA, long chain acyl-CoA; LPL, lipoprotein lipase; NEFA, nonesterified fatty acid; 14C-P, 14C-palmitate; PPAR, peroxisome proliferator-activated receptor; RG, red gastrocnemius muscle; TG, triglyceride; WAT, white adipose tissue.
Received March 1, 2004.
Accepted for publication March 25, 2004.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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activators improve insulin sensitivity and reduce adiposity. J Biol Chem 275:16638–16642 activation lowers muscle lipids and improves insulin sensitivity in high fat-fed rats: comparison with PPAR- activation. Diabetes 50:411–417 activators improve glucose homeostasis by stimulating fatty acid uptake in the adipocytes. Atherosclerosis 137(Suppl):S75–S80
and - ligand binding domain in complex with AZ 242; ligand selectivity and agonist activation in the PPAR family. Structure (Camb) 9:699–706
/ agonist with beneficial effects on insulin resistance and carbohydrate and lipid metabolism in ob/ob mice and obese Zucker rats. J Lipid Res 43:1855–1863 agonist stimulates mitochondrial fatty acid ß-oxidation in liver and skeletal muscle. Am J Physiol Endocrinol Metab 280:E270–E279
and retinoid X receptor (RXR) agonists have complementary effects on glucose and lipid metabolism in human skeletal muscle. Diabetologia 44:444–452[CrossRef][Medline]
and PPAR activators direct a distinct tissue-specific transcriptional response via a PPRE in the lipoprotein lipase gene. EMBO J 15:5336–5348[Medline]
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, P < 0.01; 
, P < 0.001 compared with HF.