A new leptin-mediated mechanism for stimulating fatty acid oxidation: a pivotal role for sarcolemmal FAT/CD36.

Leptin stimulates fatty acid oxidation in muscle and heart; but, the mechanism by which these tissues provide additional intracellular fatty acids for their oxidation remains unknown. We examined, in isolated muscle and cardiac myocytes, whether leptin, via AMP-activated protein kinase (AMPK) activation, stimulated fatty acid translocase (FAT/CD36)-mediated fatty acid uptake to enhance fatty acid oxidation. In both mouse skeletal muscle and rat cardiomyocytes, leptin increased fatty acid oxidation, an effect that was blocked when AMPK phosphorylation was inhibited by adenine 9-ß-d-arabinofuranoside or Compound C. In wild-type mice, leptin induced the translocation of FAT/CD36 to the plasma membrane and increased fatty acid uptake into giant sarcolemmal vesicles and into cardiomyocytes. In muscles of FAT/CD36-KO mice, and in cardiomyocytes in which cell surface FAT/CD36 action was blocked by sulfo-N-succinimidyl oleate, the leptin-stimulated influx of fatty acids was inhibited; concomitantly, the normal leptin-stimulated increase in fatty acid oxidation was also prevented, despite the normal leptin-induced increase in AMPK phosphorylation. Conversely, in muscle of AMPK kinase-dead mice, leptin failed to induce the translocation of FAT/CD36, along with a failure to stimulate fatty acid uptake and oxidation. Similarly, when siRNA was used to reduce AMPK in HL-1 cardiomyocytes, leptin failed to induce the translocation of FAT/CD36. Our studies have revealed a novel mechanism of leptin-induced fatty acid oxidation in muscle tissue; namely, this process is dependent on the activation of AMPK to induce the translocation of FAT/CD36 to the plasma membrane, thereby stimulating fatty acid uptake. Without increasing this leptin-stimulated, FAT/CD36-dependent fatty acid uptake process, leptin-stimulated AMPK phosphorylation does not enhance fatty acid oxidation.

Exercise- and training-induced upregulation of skeletal muscle fatty acid oxidation are not solely dependent on mitochondrial machinery and biogenesis.

Regulation of skeletal muscle fatty acid oxidation (FAO) and adaptation to exercise training have long been thought to depend on delivery of fatty acids (FAs) to muscle, their diffusion into muscle, and muscle mitochondrial content and biochemical machinery. However, FA entry into muscle occurs via a regulatable, protein-mediated mechanism, involving several transport proteins. Among these CD36 is key. Muscle contraction and pharmacological agents induce CD36 to translocate to the cell surface, a response that regulates FA transport, and hence FAO. In exercising CD36 KO mice, exercise duration (-44%), and FA transport (-41%) and oxidation (-37%) are comparably impaired, while carbohydrate metabolism is augmented. In trained CD36 KO mice, training-induced upregulation of FAO is not observed, despite normal training-induced increases in mitochondrial density and enzymes. Transfecting CD36 into sedentary WT muscle (+41%), comparable to training-induced CD36 increases (+44%) in WT muscle, markedly upregulates FAO to rates observed in trained WT mice, but without any changes in mitochondrial density and enzymes. Evidently, in vivo CD36-mediated FA transport is key for muscle fuel selection and training-induced FAO upregulation, independent of mitochondrial adaptations. This CD36 molecular mechanism challenges the view that skeletal muscle FAO is solely regulated by muscle mitochondrial content and machinery.

In vivo, fatty acid translocase (CD36) critically regulates skeletal muscle fuel selection, exercise performance, and training-induced adaptation of fatty acid oxidation.

For ~40 years it has been widely accepted that (i) the exercise-induced increase in muscle fatty acid oxidation (FAO) is dependent on the increased delivery of circulating fatty acids, and (ii) exercise training-induced FAO up-regulation is largely attributable to muscle mitochondrial biogenesis. These long standing concepts were developed prior to the recent recognition that fatty acid entry into muscle occurs via a regulatable sarcolemmal CD36-mediated mechanism. We examined the role of CD36 in muscle fuel selection under basal conditions, during a metabolic challenge (exercise), and after exercise training. We also investigated whether CD36 overexpression, independent of mitochondrial changes, mimicked exercise training-induced FAO up-regulation. Under basal conditions CD36-KO versus WT mice displayed reduced fatty acid transport (-21%) and oxidation (-25%), intramuscular lipids (less than or equal to -31%), and hepatic glycogen (-20%); but muscle glycogen, VO(2max), and mitochondrial content and enzymes did not differ. In acutely exercised (78% VO(2max)) CD36-KO mice, fatty acid transport (-41%), oxidation (-37%), and exercise duration (-44%) were reduced, whereas muscle and hepatic glycogen depletions were accelerated by 27-55%, revealing 2-fold greater carbohydrate use. Exercise training increased mtDNA and ß-hydroxyacyl-CoA dehydrogenase similarly in WT and CD36-KO muscles, but FAO was increased only in WT muscle (+90%). Comparable CD36 increases, induced by exercise training (+44%) or by CD36 overexpression (+41%), increased FAO similarly (84-90%), either when mitochondrial biogenesis and FAO enzymes were up-regulated (exercise training) or when these were unaltered (CD36 overexpression). Thus, sarcolemmal CD36 has a key role in muscle fuel selection, exercise performance, and training-induced muscle FAO adaptation, challenging long held views of mechanisms involved in acute and adaptive regulation of muscle FAO.

Migration- and exercise-induced changes to flight muscle size in migratory birds and association with IGF1 and myostatin mRNA expression.

Seasonal adjustments to muscle size in migratory birds may result from preparatory physiological changes or responses to changed workloads. The mechanisms controlling these changes in size are poorly understood. We investigated some potential mediators of flight muscle size (myostatin and insulin-like growth factor, IGF1) in pectoralis muscles of wild wintering or migrating white-throated sparrows (Zonotrichia albicollis), captive white-throated sparrows that were photoperiod manipulated to be in a `wintering' or `migratory' (Zugunruhe) state, and captive European starlings (Sturnus vulgaris) that were either exercised for 2 weeks in a wind tunnel or untrained. Flight muscle size increased in photo-stimulated `migrants' and in exercised starlings. Acute exercise but not long-term training caused increased expression of IGF1, but neither caused a change in expression of myostatin or its metalloprotease activator TLL1. Photo-stimulated `migrant' sparrows demonstrated increased expression of both myostatin and IGF1, but wild sparrows exhibited no significant seasonal changes in expression of either myostatin or IGF1. Additionally, in both study species we describe several splice variants of myostatin that are shared with distantly related bird species. We demonstrate that their expression patterns are not different from those of the typical myostatin, suggesting that they have no functional importance and may be mistakes of the splicing machinery. We conclude that IGF1 is likely to be an important mediator of muscle phenotypic flexibility during acute exercise and during endogenous, seasonal preparation for migration. The role of myostatin is less clear, but its paradoxical increase in photo-stimulated `migrants' may indicate a role in seasonal adjustments of protein turnover.

Nuclear SIRT1 activity, but not protein content, regulates mitochondrial biogenesis in rat and human skeletal muscle.

Silent mating type information regulator 2 homolog 1 (SIRT1)-mediated peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) deacetylation is potentially key for activating mitochondrial biogenesis. Yet, at the whole muscle level, SIRT1 is not associated with mitochondrial biogenesis (Gurd, BJ, Yoshida Y, Lally J, Holloway GP, Bonen A. J Physiol 587: 1817-1828, 2009). Therefore, we examined nuclear SIRT1 protein and activity in muscle with varied mitochondrial content and in response to acute exercise. We also measured these parameters after stimulating mitochondrial biogenesis with chronic muscle contraction and 5-aminoimidazole-4-carboxamide-1-ß-d-ribofuranoside (AICAR) administration in rodents and exercise training in humans. In skeletal and heart muscles, nuclear SIRT1 protein was negatively correlated with indices of mitochondrial density (citrate synthase activity, CS; cytochrome oxidase IV, COX IV), but SIRT1 activity was positively correlated with these parameters (r > 0.98). Acute exercise did not alter nuclear SIRT1 protein but did induce a time-dependent increase in nuclear SIRT1 activity. This increase in SIRT1 activity was temporally related to increases in mRNA expression of genes activated by PGC-1α. Both chronic muscle stimulation and AICAR increased mitochondrial biogenesis and muscle PGC-1α, but not nuclear PGC-1α. Concomitantly, muscle and nuclear SIRT1 protein contents were reduced, but nuclear SIRT1 activity was increased. In human muscle, training-induced mitochondrial biogenesis did not alter muscle or nuclear SIRT1 protein content, but it did increase muscle and nuclear PGC-1α and SIRT1 activity. Thus, nuclear SIRT1 activity, but not muscle or nuclear SIRT1 protein content, is associated with contraction-stimulated mitochondrial biogenesis in rat and human muscle, possibly via AMPK activation.

Preparing for migration? The effects of photoperiod and exercise on muscle oxidative enzymes, lipid transporters, and phospholipids in white-crowned sparrows.

The extreme energetic demands of avian migration result in various physiological changes that can be observed during the migratory period. However, the degree to which birds alter muscle physiology in advance of migration has been poorly studied. We studied the effects of "migratory" photoperiod and exercise on metabolic enzymes, fatty acid transporter mRNA expression, and muscle phospholipid fatty acid composition in captive white-crowned sparrows (Zonotrichia leucophrys). Ten sparrows were held on short photoperiod (8L:16D) for 58 d then switched to long days (16L:8D) for 3 wk before sampling. Increased nightly activity indicated that the birds were indeed in migratory condition. Another 13 birds were held on short days during the entire experiment, and a subset (5) were exercised for 1 h every other day for the last 2 wk. "Migratory" photoperiod did not change the activities of citrate synthase, carnitine palmitoyl transferase, and 3-hydroxyacyl-CoA dehydrogenase or the expression of FAT/CD36, FABPpm, and H-FABP mRNA in pectoralis muscle, suggesting that these cannot be increased in advance of migratory flight. Docosahexaenoic acid increased in pectoralis muscle phospholipids with exercise but was negatively correlated with catabolic enzyme activity, indicating that the presence of this fatty acid may not aid migratory performance as suggested by other studies.

Seasonal upregulation of fatty acid transporters in flight muscles of migratory white-throated sparrows (Zonotrichia albicollis).

Endurance flights of birds, some known to last several days, can only be sustained by high rates of fatty acid uptake by flight muscles. Previous research in migratory shorebirds indicates that this is made possible in part by very high concentrations of cytosolic heart-type fatty acid binding protein (H-FABP), which is substantially upregulated during migratory seasons. We investigated if H-FABP and other components of muscle fatty acid transport also increase during these seasons in a passerine species, the white-throated sparrow (Zonotrichia albicollis). Fatty acid translocase (FAT/CD36) and plasma-membrane fatty acid binding protein (FABPpm) are well characterized mammalian proteins that facilitate transport of fatty acid through the muscle membrane, and in this study they were identified for the first time in birds. We used quantitative PCR to measure mRNA of FAT/CD36, FABPpm and H-FABP and immunoblotting to measure protein expression of FABPpm and H-FABP in the pectoralis muscles of sparrows captured in migratory (spring, fall) and non-migratory (winter) seasons. During migratory seasons, mRNA expression of these genes increased 70-1000% above wintering levels, while protein expression of H-FABP and FABPpm increased 43% and 110% above wintering levels. Activities of key metabolic enzymes, 3-hydroxyacyl-CoA-dehydrogenase (HOAD), carnitine palmitoyl transferase II (CPT II), and citrate synthase (CS) also increased (90-110%) in pectoralis muscles of migrant birds. These results support the hypothesis that enhanced protein-mediated transport of fatty acids from the circulation into muscle is a key component of the changes in muscle biochemistry required for migration in birds.