Fenofibrate-Induced Muscular Toxicity Is Associated with a Metabolic Shift Limited to Type-1 Muscles in Rats

Abstract

Morphological changes and mRNA expression levels in type-1 predominant soleus and type-2 predominant tensor fasciae latae muscles of rats treated with fenofibrate were investigated. After fenofibrate by oral gavage at 300 mg/kg/day for 28 days, degeneration/necrosis and regeneration of muscle fibers, cellular infiltration, and fibrosis were seen in soleus muscle. Additionally, expression of PDK4, CPT1-M, CPT2, and FACO mRNAs was increased. In contrast, no morphological changes or mRNA induction were apparent in tensor fasciae latae muscle. These data suggest that sensitivity to fenofibrate-induced muscle toxicity differs among muscles, with only type-1 fibers being susceptible. The up-regulation of PDK4, CPTs and FACO mRNA expression in soleus muscle indicates that the energy source is switched from glucose to fatty acids, and this might be related to the observed fenofibrate-induced muscular toxicity.

Introduction

Fibrates are widely used in the clinic for their triglyceride (TG)-lowering properties. They are effective at lowering elevated plasma TGs by activation of the nuclear receptor, peroxisome proliferator-activated receptor-α (PPARα) in hepatocytes (Brunmair et al. 2004; Matzno et al. 2006; Staels et al. 1998), inducing expression of genes involved in intracellular fatty acid metabolism, such as mitochondrial β-oxidation (Matzno et al. 2006; Staels et al. 1998). Fibrates are generally considered to be safe and well tolerated (Johnson et al. 2005), but they may cause myopathy and rhabdomyolysis in humans (Hodel 2002; Lane and Mastaglia 1978; Warren, Blumbergs, and Thompson 2002). Only few details have been reported regarding the myopathy induced by fibrates in animals (Afifi et al. 1984; Teravainen, Larsen, and Hillbom 1977).

Skeletal muscle fibers are variously classified on the basis of morphological, biochemical, and physiologic properties (Herbison, Jaweed, and Ditunno 1982; Jones, Mohr, and Hunt 1991; Talmadge, Roy, and Edgerton 1993). We previously reported that sensitivity to clofibrate-induced muscle toxicity differs among muscles, with type-1 fibers being susceptible (Okada et al. 2007). In the skeletal muscle, glucose and lipids are utilized as substrates for energy production (Russell et al. 2003; Samec et al. 2002; Watt, Heigenhauser, and Spriet 2002), regulated by the pyruvate dehydrogenase (PDH) complex and the carnitine palmitoyltransferase (CPT) system, respectively (Sugden and Holness 1994).

In the present study, we therefore investigated glucose and lipid metabolism-related gene expression as well as histological changes in rat skeletal muscles induced by fenofibrate. For this purpose, type-1 predominant soleus (87% ± 4% slow twitch oxidative fibers) and type-2 predominant tensor fasciae latae (89% ± 5% fast twitch glycolytic fibers) were selected for examination (Armstrong and Phelps 1984).

Materials and Methods

Animals

Male Crl:CD(SD) rats aged 5 weeks were purchased from Charles River Laboratories Japan, Inc. (Yokohama, Japan) and acclimatized for 1 week in an air-conditioned animal room at 22°C with a 12-hr light/dark cycle. The animals were given CRF-1 (Oriental Yeast Co., Ltd., Tokyo, Japan) and tap water ad libitum.

Dosing

Six rats were treated with fenofibrate (Sigma-Aldrich, St. Louis, MO, USA) once daily by oral gavage at 300 mg/kg for 28 days. Fenofibrate was suspended at 60 mg/mL in a 0.5% hydroxypropyl methylcellulose (HPMC, Shin-Etsu Chemical Co. Ltd., Tokyo, Japan). Six additional rats were similarly given 0.5% HPMC for 28 days as controls.

Necropsy and Tissue Collection

On day 29, rats were sacrificed by exsanguination by cutting the abdominal aorta under ether anesthesia after blood sampling for blood chemistry. Soleus and tensor fasciae latae muscles of both hindlimbs were removed. Those from the right side were fixed in 10% neutral buffered formalin, and those from the left were immersed in RNA later^™ RNA Stabilization Regent (QUIAGEN^®, Valencia, CA, USA).

Light Microscopy

Formalin-fixed soleus and tensor fasciae latae muscles were routinely embedded in paraffin, sectioned, and stained with hematoxylin and eosin (HE).

Quantitative Real-Time Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR)

To examine the expression of PDK4, PDC, FACO, CPT1-M, CPT1-L, CPT2, and β-actin, total RNA was extracted from soleus and tensor fasciae latae muscles using ISOGEN (Nippon Gene, Tokyo, Japan) according to the manufacturer’s instruction. Quantitative real-time RT-PCR with a QuantiTect SYBR Green RT-PCR Kit (QIAGEN^®, Valencia, CA, USA) was performed using the ABI PRISM 7700 Sequence Detection System (Applied Biosystems, Foster City, CA, USA). The expression of genes was calculated as relative differences between control and treatment groups. The sequences of the primers used for real-time RT-PCR analysis are shown in Table 1.

Statistical Evaluation

The data from blood chemistry, body weight, and real-time RT-PCR were statistically analyzed by Dunnett’s multiple comparisons. A p value of less than .05 was considered to be statistically significant.

All experiments were performed according to the “Rules for Feeding and Storage of Experimental Animals and Animal Experiments” and approved by the Institutional Animal Care and Use Committee of Mitsubishi Tanabe Pharma Corporation from the point of view of animal welfare.

Results

Blood Chemistry and Body Weight

The values of serum TG and glucose and body weight are summarized in Table 2. Decrease in serum TG levels was noted in rats treated with fenofibrate for 28 days. The value of serum glucose and body weight was not changed.

Histopathological Examination

The incidence of microscopic changes in soleus muscle is summarized in Table 3. In soleus muscle, fenofibrate-related microscopic changes were noted in rats treated for 28 days. In contrast, no change was seen in the tensor fasciae latae muscle. The lesions observed in soleus muscle were degeneration/ necrosis of muscle fibers, fiber regeneration, infiltration of leukocytes, and fibrosis (Figure 1).

Analysis of Fenofibrate-Induced Gene Expression

Changes in the mRNA levels for PDK4, FACO, CPT1-M, and CPT2 in soleus and tensor fasciae latae muscles are shown in Figures 2 and 3, respectively.

In fenofibrate-treated rats for 28 days, obvious increase of PDK4 mRNA expression and a slight increase of FACO, CPT1-M, and CPT2 were observed only in soleus muscle with some muscular lesions. In contrast, no mRNA induction was apparent in tensor fasciae latae muscle. Changes in mRNA levels for PDC and CPT1-L were not evident in either soleus or tensor fasciae latae muscles.

Discussion

In the present study, fenofibrate was found to induce lesions only in type-1 predominant soleus muscle, with no morphological changes being observed in type-2 predominant tensor fasciae latae muscle. These data indicate that the sensitivity to fenofibrate-induced toxicity differs among muscles, in line with our previous report that clofibrate, a PPARα agonist, induces lesions limited to type-1 predominant soleus muscle and type-1 muscle fibers in diaphragm of rats (Okada et al. 2007). From these results, type-1 muscle fibers appear to be targets of fibrate-induced muscular toxicity.

Induction of glycolysis-related gene expression was also limited to type-1 predominant soleus muscle in the present study. PDK4 is one component of the pyruvate dehydrogenase (PDH) complex that regulates glucose oxidation (Sugden and Holness 1994). It phosphorylates and inactivates PDC, which catalyzes irreversible decarboxylation of pyruvate to acetyl-CoA (Holness et al. 2002; Pilegaard and Neufer 2004; Sugden 2003; Sugden and Holness 1994). Motojima (2002) reported that WY14,643 induced up-regulation of PDK4 expression in various tissues including the liver, skeletal muscle, and adipose tissue in mice. Up-regulation of PDK4 mRNA expression limits glucose oxidation, enhances fatty acid utilization, and leads to a reduction of serum TG (Holness et al. 2002; Motojima 2002; Motojima and Seto 2003). In the present study, PDK4 mRNA induction in the soleus muscle was observed and the value of serum TG decreased while no change was noted in serum glucose level. Therefore, limitation of glucose utilization in the muscle may be one cause of fenofibrate-induced myopathy.

Fenofibrate also induced gene expression of fatty acid β-oxidation only in type-1 predominant soleus muscle. CPTs and FACO commit fatty acids to β-oxidation, being located in mitochondria and peroxisomes, respectively (Minnich et al. 2001; Nanji et al. 2004; Sugden and Holness 1994). In this study, the expression of CPT-1M, CPT2, and FACO mRNAs was increased only in type-1 muscle. Therefore, we can conclude that fenofibrate stimulates mitochondrial and peroxisomal fatty acid β-oxidation only in this muscle type.

In conclusion, fenofibrate-induced myopathy and glucose and lipid metabolism-related mRNAs induction differ among muscles, with type-1 predominant soleus muscle being susceptible. Up-regulation of particular genes suggests that the energy fuel is shifted from glucose to fatty acid and that such metabolic switching might be one cause of muscular toxicity induced by fibrates.