ABSTRACT
In skeletal muscle the Rab-GTPase-activating protein TBC1D1 has been implicated in the regulation of fatty acid oxidation by an unknown mechanism. We determined whether TBC1D1 altered fatty acid utilization via changes in protein-mediated fatty acid transport and/or selected enzymes regulating mitochondrial fatty acid oxidation. We also determined the effects of TBC1D1 on glucose transport and oxidation. Electrotransfection of mouse soleus muscles with TBC1D1 cDNA increased TBC1D1 protein after 2 wk (P<0.05), without altering its paralog AS160. TBC1D1 overexpression decreased basal palmitate oxidation (-22%) while blunting 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR)-stimulated palmitate oxidation (-18%). There was a tendency to increase fatty acid esterification (+10 nmol·g(-1)·60 min(-1), P=0.07), which reflected the reduction in fatty acid oxidation (-12 nmol·g(-1)·60 min(-1)). Concomitantly, basal (+21%) and AICAR-stimulated glucose oxidation (+8%) were increased in TBC1D1-transfected muscles relative to their respective controls (P<0.05), independent of changes in GLUT4 and glucose transport. The reductions in TBC1D1-mediated fatty acid oxidation could not be attributed to changes in the transporter FAT/CD36, muscle mitochondrial content, CPT1 expression or the expression and phosphorylation of AS160, acetyl-CoA carboxylase, or AMPK. However, TBC1D1 overexpression reduced ß-HAD enzyme activity (-18%, P<0.05). In conclusion, TBC1D1-mediated reduction of muscle fatty acid oxidation appears to occur via inhibition of ß-HAD activity.
Subject(s)
3-Hydroxyacyl CoA Dehydrogenases/metabolism , Muscle, Skeletal/enzymology , Nuclear Proteins/metabolism , Palmitates/metabolism , 3-Hydroxyacyl CoA Dehydrogenases/genetics , Aminoimidazole Carboxamide/analogs & derivatives , Animals , Fatty Acids/metabolism , GTPase-Activating Proteins , Gene Expression Regulation/physiology , Glucose/metabolism , Lipid Peroxidation , Mice , Mice, Inbred C57BL , Nuclear Proteins/genetics , Oxidation-Reduction , RibonucleotidesABSTRACT
AIM: The increase in skeletal muscle fatty acid metabolism during exercise has been associated with the release of calcium. We examined whether this increase in fatty acid oxidation was attributable to a calcium-induced translocation of the fatty acid transporter CD36 to the sarcolemma, thereby providing an enhanced influx of fatty acids to increase their oxidation. METHODS: Calcium release was triggered by caffeine (3 mm) to examine fatty acid oxidation in intact soleus muscles of WT and CD36-KO mice, while fatty acid transport and mitochondrial fatty acid oxidation were examined in giant vesicles and isolated mitochondria, respectively, from caffeine-perfused hindlimb muscles of WT and CD36-KO mice. Western blotting was used to examine calcium-induced signalling. RESULTS: In WT, caffeine stimulated muscle palmitate oxidation (+136%), but this was blunted in CD36-KO mice (-70%). Dantrolene inhibited (WT) or abolished (CD36-KO) caffeine-induced palmitate oxidation. In muscle, caffeine-stimulated palmitate oxidation was not attributable to altered mitochondrial palmitate oxidation. Instead, in WT, caffeine increased palmitate transport (+55%) and the translocation of fatty acid transporters CD36, FABPpm, FATP1 and FATP4 (26-70%) to the sarcolemma. In CD36-KO mice, caffeine-stimulated FABPpm, and FATP1 and 4 translocations were normal, but palmitate transport was blunted (-70%), comparable to the reductions in muscle palmitate oxidation. Caffeine did not alter the calcium-/calmodulin-dependent protein kinase II phosphorylation but did increase the phosphorylation of AMPK and acetyl-CoA carboxylase comparably in WT and CD36-KO. CONCLUSION: These studies indicate that sarcolemmal CD36-mediated fatty acid transport is a primary mediator of the calcium-induced increase in muscle fatty acid oxidation.
