Fatty acid binding protein facilitates sarcolemmal fatty acid transport but not mitochondrial oxidation in rat and human skeletal muscle

Abstract

The transport of long-chain fatty acids (LCFAs) across mitochondrial membranes is regulated by carnitine palmitoyltransferase I (CPTI) activity. However, it appears that additional fatty acid transport proteins, such as fatty acid translocase (FAT)/CD36, influence not only LCFA transport across the plasma membrane, but also LCFA transport into mitochondria. Plasma membrane-associated fatty acid binding protein (FABPpm) is also known to be involved in sacrolemmal LCFA transport, and it is also present on the mitochondria. At this location, it has been identified as mitochondrial aspartate amino transferase (mAspAT), despite being structurally identical to FABPpm. Whether this protein is also involved in mitochondrial LCFA transport and oxidation remains unknown. Therefore, we have examined the ability of FABPpm/mAspAT to alter mitochondrial fatty acid oxidation. Muscle contraction increased (P < 0.05) the mitochondrial FAT/CD36 content in rat (+22%) and human skeletal muscle (+33%). By contrast, muscle contraction did not alter the content of mitochondrial FABPpm/mAspAT protein in either rat or human muscles. Electrotransfecting rat soleus muscles, in vivo, with FABPpm cDNA increased FABPpm protein in whole muscle (+150%; P < 0.05), at the plasma membrane (+117%; P < 0.05) and in mitochondria (+80%; P < 0.05). In these FABPpm-transfected muscles, palmitate transport into giant vesicles was increased by +73% (P < 0.05), and fatty acid oxidation in intact muscle was increased by +18% (P < 0.05). By contrast, despite the marked increase in mitochondrial FABPpm/mAspAT protein content (+80%), the rate of mitochondrial palmitate oxidation was not altered (P > 0.05). However, electrotransfection increased mAspAT activity by +70% (P < 0.05), and the mitochondrial FABPpm/mAspAT protein content was significantly correlated with mAspAT activity (r = 0.75). It is concluded that FABPpm has two distinct functions depending on its subcellular location: (a) it contributes to increasing sarcolemmal LCFA transport while not contributing directly to LCFA transport into mitochondria; and (b) its primary role at the mitochondria level is to transport reducing equivalents into the matrix.

Figures

Figure 1. Effects of muscle contraction on the content of mitochondrial FABPpm/mAspAT and FAT/CD36

Values are means ±s.e.m., expressed in arbitrary units. A, mitochondrial FABPpm/mAspAT content in rat soleus muscle at rest, and following 30 min of electrical stimulation (n= 5). B, mitochondrial FABPpm/mAspAT content in human vastus lateralis muscle at rest, and following 2 h of cycling at ∼60% peak O2 consumption rate (n= 12). C, mitochondrial FAT/CD36 content in rat soleus muscle at rest, and following 30 min of electrical stimulation (n= 5). D, mitochondrial FAT/CD36 content in human vastus lateralis muscle at rest, and following 2 h of cycling at ∼60% (n= 7). *Significantly different (P < 0.05) from rest.

Figure 2. Effects of electrotransfection with FABPpm cDNA on soleus muscle

Values are means ±s.e.m.; protein contents expressed in arbitrary units, and transport expressed in pmol (mg wet weight)−1 15 s−1 (n= 12). A, whole-muscle FABPpm protein content. B, plasmalemmal FABPpm protein content. C, palmitate transport into giant sarcolemmal vesicles. ww, wet weight. *Significantly different (P < 0.05) from control.

Figure 3. Effects of electrotransfection with FABPpm cDNA on the fate of palmitate in soleus muscle

Values are means ±s.e.m., expressed in nmol mg wet weight−1 hour−1 (n= 12). A, palmitate incorporation into the diacylglycerol pool. B, palmitate incorporation into triacylglycerol pool. C, palmitate oxidation. ww, wet weight. *Significantly different (P < 0.05) from control.

Figure 4. Representative Western blots performed on mitochondria isolated from control and electrotransfected solei

Figure 5. Effects of electrotransfection with FABPpm cDNA on the content of soleus muscle mitochondrial proteins, and the ability of mitochondria to oxidize palmitate

Values are means ±s.e.m., expressed in arbitrary units (n= 5). A, mitochondrial FABPpm/mAspAT protein content. B, mitochondrial FAT/CD36 protein content. C, Cox-IV protein content. D, isolated mitochondrial palmitate oxidation, expressed in nmol mg protein−1 hour−1. *Significantly different (P < 0.05) from control.

Figure 6. Effects of electrotransfection with FABPpm cDNA on mitochondrial enzymatic activity

Values are means ±s.e.m., expressed in μmol min−1 g wet weight−1. A, mitochondrial aspartate aminotransferase (mAspAT) activity in homogenates (n= 10). B, mitochondrial aspartate aminotransferase activity (mAspAT) in isolated mitochondria (n= 5). C, homogenate β-hydroxyacyl-CoA dehydrogenase (β-HAD) activity (n= 10). D, homogenate citrate synthase (CS) activity (n= 10). ww, wet weight. *Significantly different (P < 0.05) from control.

Figure 7. Pearson correlation calculated between mitochondrial FABPpm/mAspAT content and mAspAT activity in homogenates

n= 20 (10 control and 10 transfected) muscles.