Arend Bonen

Membrane Fatty Acid Transporters as Regulators of Lipid Metabolism: Implications for Metabolic Disease

Long-chain fatty acids and lipids serve a wide variety of functions in mammalian homeostasis, particularly in the formation and dynamic properties of biological membranes and as fuels for energy production in tissues such as heart and skeletal muscle. On the other hand, long-chain fatty acid metabolites may exert toxic effects on cellular functions and cause cell injury. Therefore, fatty acid uptake into the cell and intracellular handling need to be carefully controlled. In the last few years, our knowledge of the regulation of cellular fatty acid uptake has dramatically increased. Notably, fatty acid uptake was found to occur by a mechanism that resembles that of cellular glucose uptake. Thus, following an acute stimulus, particularly insulin or muscle contraction, specific fatty acid transporters translocate from intracellular stores to the plasma membrane to facilitate fatty acid uptake, just as these same stimuli recruit glucose transporters to increase glucose uptake. This regulatory mechanism is important to clear lipids from the circulation postprandially and to rapidly facilitate substrate provision when the metabolic demands of heart and muscle are increased by contractile activity. Studies in both humans and animal models have implicated fatty acid transporters in the pathogenesis of diseases such as the progression of obesity to insulin resistance and type 2 diabetes. As a result, membrane fatty acid transporters are now being regarded as a promising therapeutic target to redirect lipid fluxes in the body in an organ-specific fashion.

Triacylglycerol accumulation in human obesity and type 2 diabetes is associated with increased rates of skeletal muscle fatty acid transport and increased sarcolemmal FAT/CD36

We examined whether, in human obesity and type 2 diabetes, long chain fatty acid (LCFA) transport into skeletal muscle is upregulated and contributes to an excess intramuscular triacylglycerol accumulation. In giant sarcolemmal vesicles prepared from human skeletal muscle, LCFA transport rates were upregulated ~4‐fold and were associated with an increased intramuscular triacylglycerol content in obese individuals and in type 2 diabetics. In these individuals, the increased sarcolemmal LCFA transport rate was not associated with an altered expression of FAT/CD36 or FABPpm. Instead, the increase in the LCFA transport rate was associated with an increase in sarcolemmal FAT/CD36 but not sarcolemmal FABPpm. Rates of fatty acid esterification were increased threefold in isolated human muscle strips obtained from obese subjects, while concomitantly rates of fatty acid oxidation were not altered. Thus, the increased rate of fatty acid transport may contribute to the increased rates of triacylglycerol accumulation in human skeletal muscle. The altered FAT/CD36 trafficking in muscle from obese subjects and type 2 diabetics juxtaposes the known alterations in GLUT4 trafficking, i.e., GLUT4 is known to be retained in its intracellular depots while FAT/CD36 is retained at the sarcolemma. This redistribution of FAT/CD36 to the sarcolemma may contribute to the etiology of insulin resistance in human muscle, and hence, FAT/CD36 provides another potential therapeutic target for the prevention and/or treatment of insulin resistance.

Lactic Acid Efflux from White Skeletal Muscle Is Catalyzed by the Monocarboxylate Transporter Isoform MCT3

The newly cloned proton-linked monocarboxylate transporter MCT3 was shown by Western blotting and immunofluorescence confocal microscopy to be expressed in all muscle fibers. In contrast, MCT1 is expressed most abundantly in oxidative fibers but is almost totally absent in fast-twitch glycolytic fibers. Thus MCT3 appears to be the major MCT isoform responsible for efflux of glycolytically derived lactic acid from white skeletal muscle. MCT3 is also expressed in several other tissues requiring rapid lactic acid efflux. The expression of both MCT3 and MCT1 was decreased by 40–60% 3 weeks after denervation of rat hind limb muscles, whereas chronic stimulation of the muscles for 7 days increased expression of MCT1 2–3-fold but had no effect on MCT3 expression. The kinetics and substrate and inhibitor specificities of monocarboxylate transport into cell lines expressing only MCT3 or MCT1 have been determined. Differences in the properties of MCT1 and MCT3 are relatively modest, suggesting that the significance of the two isoforms may be related to their regulation rather than their intrinsic properties.

Repeated transient mRNA bursts precede increases in transcriptional and mitochondrial proteins during training in human skeletal muscle

Exercise training induces mitochondrial biogenesis, but the time course of molecular sequelae that accompany repetitive training stimuli remains to be determined in human skeletal muscle. Therefore, throughout a seven‐session, high‐intensity interval training period that increased (12%), we examined the time course of responses of (a) mitochondrial biogenesis and fusion and fission proteins, and (b) selected transcriptional and mitochondrial mRNAs and proteins in human muscle. Muscle biopsies were obtained 4 and 24 h after the 1st, 3rd, 5th and 7th training session. PGC‐1α mRNA was increased >10‐fold 4 h after the 1st session and returned to control within 24 h. This ‘saw‐tooth’ pattern continued until the 7th bout, with smaller increases after each bout. In contrast, PGC‐1α protein was increased 24 h after the 1st bout (23%) and plateaued at +30–40% between the 3rd and 7th bout. Increases in PGC‐1β mRNA and protein were more delayed and smaller, and did not persist. Distinct patterns of increases were observed in peroxisome proliferator‐activated receptor (PPAR) α and γ protein (1 session), PPAR β/δ mRNA and protein (5 sessions) and nuclear respiratory factor‐2 protein (3 sessions) while no changes occurred in mitochondrial transcription factor A protein. Citrate synthase (CS) and β‐HAD mRNA were rapidly increased (1 session), followed 2 sessions later (session 3) by increases in CS and β‐HAD activities, and mitochondrial DNA. Changes in COX‐IV mRNA (session 3) and protein (session 5) were more delayed. Training also increased mitochondrial fission proteins (fission protein‐1, >2‐fold; dynamin‐related protein‐1, 47%) and the fusion protein mitofusin‐1 (35%) but not mitofusin‐2. This study has provided the following novel information: (a) the training‐induced increases in transcriptional and mitochondrial proteins appear to result from the cumulative effects of transient bursts in their mRNAs, (b) training‐induced mitochondrial biogenesis appears to involve re‐modelling in addition to increased mitochondrial content, and (c) the ‘transcriptional capacity’ of human muscle is extremely sensitive, being activated by one training bout.

Do more constraints mean less leisure? Examining the relationship between constraints and participation.

Underlying much of the literature on leisure constraints is the assumption that the constraints that people report do, in fact, act as barriers to participation. However, to date, there has been a ...

Defective fatty acid uptake modulates insulin responsiveness and metabolic responses to diet in CD36-null mice

Deficiency of the membrane protein FAT/CD36 causes a marked defect in fatty acid uptake by various tissues and is genetically linked to insulin resistance in rats and humans. Here, we examined insulin responsiveness of CD36-/- mice. When fed a diet high in complex carbohydrates and low (5%) in fat, these animals cleared glucose faster than the wild-type. In vivo, uptake of 2-fluorodeoxyglucose by muscle was increased severalfold, and in vitro, insulin responsiveness of glycogenesis by the soleus was enhanced. Null mice had lower glycogen levels in muscle and liver, lower muscle triglyceride levels, and increased liver triglyceride content--all findings consistent with increased insulin-sensitivity. However, when the chow diet was switched to one high in fructose, CD36-/- mice but not wild-type mice developed marked glucose intolerance, hyperinsulinemia, and decreased muscle glucose uptake. High-fat diets impaired glucose tolerance equally in both groups, although CD36 deficiency helped moderate insulin-responsive muscle glucose oxidation. In conclusion, CD36 deficiency enhances insulin responsiveness on a high-starch, low-fat diet. It predisposes to insulin resistance induced by high fructose and partially protects from that induced by high-fat diets. In humans, CD36 deficiency may be an important factor in the metabolic adaptation to diet and in susceptibility to some forms of diet-induced pathology.

Palmitate transport and fatty acid transporters in red and white muscles

We performed studies 1) to investigate the kinetics of palmitate transport into giant sarcolemmal vesicles, 2) to determine whether the transport capacity is greater in red muscles than in white muscles, and 3) to determine whether putative long-chain fatty acid (LCFA) transporters are more abundant in red than in white muscles. For these studies we used giant sarcolemmal vesicles, which contained cytoplasmic fatty acid binding protein (FABPc), an intravesicular fatty acid sink. Intravesicular FABPcconcentrations were sufficiently high so as not to limit the uptake of palmitate under conditions of maximal palmitate uptake (i.e., 4.5-fold excess in white and 31.3-fold excess in red muscle vesicles). All of the palmitate taken up was recovered as unesterified palmitate. Palmitate uptake was reduced by phloretin (-50%), sulfo- N-succinimidyl oleate (-43%), anti-plasma membrane-bound FABP (FABPpm, -30%), trypsin (-45%), and when incubation temperature was lowered to 0°C (-70%). Palmitate uptake was also reduced by excess oleate (-65%), but not by excess octanoate or by glucose. Kinetic studies showed that maximal transport was 1.8-fold greater in red vesicles than in white vesicles. The Michaelis-Menten constant in both types of vesicles was ∼6 nM. Fatty acid transport protein mRNA and fatty acid translocase (FAT) mRNA were about fivefold greater in red muscles than in white muscles. FAT/CD36 and FABPpm proteins in red vesicles or in homogenates were greater than in white vesicles or homogenates ( P < 0.05). These studies provide the first evidence of a protein-mediated LCFA transport system in skeletal muscle. In this tissue, palmitate transport rates are greater in red than in white muscles because more LCFA transporters are available.We performed studies 1) to investigate the kinetics of palmitate transport into giant sarcolemmal vesicles, 2) to determine whether the transport capacity is greater in red muscles than in white muscles, and 3) to determine whether putative long-chain fatty acid (LCFA) transporters are more abundant in red than in white muscles. For these studies we used giant sarcolemmal vesicles, which contained cytoplasmic fatty acid binding protein (FABPc), an intravesicular fatty acid sink. Intravesicular FABPc concentrations were sufficiently high so as not to limit the uptake of palmitate under conditions of maximal palmitate uptake (i.e., 4.5-fold excess in white and 31.3-fold excess in red muscle vesicles). All of the palmitate taken up was recovered as unesterified palmitate. Palmitate uptake was reduced by phloretin (-50%), sulfo-N-succinimidyl oleate (-43%), anti-plasma membrane-bound FABP (FABPpm, -30%), trypsin (-45%), and when incubation temperature was lowered to 0 degrees C (-70%). Palmitate uptake was also reduced by excess oleate (-65%), but not by excess octanoate or by glucose. Kinetic studies showed that maximal transport was 1.8-fold greater in red vesicles than in white vesicles. The Michaelis-Menten constant in both types of vesicles was approximately 6 nM. Fatty acid transport protein mRNA and fatty acid translocase (FAT) mRNA were about fivefold greater in red muscles than in white muscles. FAT/CD36 and FABPpm proteins in red vesicles or in homogenates were greater than in white vesicles or homogenates (P < 0.05). These studies provide the first evidence of a protein-mediated LCFA transport system in skeletal muscle. In this tissue, palmitate transport rates are greater in red than in white muscles because more LCFA transporters are available.

Modest PGC-1α Overexpression in Muscle in Vivo Is Sufficient to Increase Insulin Sensitivity and Palmitate Oxidation in Subsarcolemmal, Not Intermyofibrillar, Mitochondria

PGC-1α overexpression in skeletal muscle, in vivo, has yielded disappointing and unexpected effects, including disrupted cellular integrity and insulin resistance. These unanticipated results may stem from an excessive PGC-1α overexpression in transgenic animals. Therefore, we examined the effects of a modest PGC-1α overexpression in a single rat muscle, in vivo, on fuel-handling proteins and insulin sensitivity. We also examined whether modest PGC-1α overexpression selectively targeted subsarcolemmal (SS) mitochondrial proteins and fatty acid oxidation, because SS mitochondria are metabolically more plastic than intermyofibrillar (IMF) mitochondria. Among metabolically heterogeneous rat hindlimb muscles, PGC-1α was highly correlated with their oxidative fiber content and with substrate transport proteins (GLUT4, FABPpm, and FAT/CD36) and mitochondrial proteins (COXIV and mTFA) but not with insulin-signaling proteins (phosphatidylinositol 3-kinase, IRS-1, and Akt2), nor with 5′-AMP-activated protein kinase, α2 subunit, and HSL. Transfection of PGC-1α into the red (RTA) and white tibialis anterior (WTA) compartments of the tibialis anterior muscle increased PGC-1α protein by 23-25%. This also induced the up-regulation of transport proteins (FAT/CD36, 35-195%; GLUT4, 20-32%) and 5′-AMP-activated protein kinase, α2 subunit (37-48%), but not other proteins (FABPpm, IRS-1, phosphatidylinositol 3-kinase, Akt2, and HSL). SS and IMF mitochondrial proteins were also up-regulated, including COXIV (15-75%), FAT/CD36 (17-30%), and mTFA (15-85%). PGC-1α overexpression also increased palmitate oxidation in SS (RTA, +116%; WTA, +40%) but not in IMF mitochondria, and increased insulin-stimulated phosphorylation of AKT2 (28-43%) and rates of glucose transport (RTA, +20%; WTA, +38%). Thus, in skeletal muscle in vivo, a modest PGC-1α overexpression up-regulated selected plasmalemmal and mitochondrial fuel-handling proteins, increased SS (not IMF) mitochondrial fatty acid oxidation, and improved insulin sensitivity.

PGC-1α regulation by exercise training and its influences on muscle function and insulin sensitivity

The peroxisome proliferator-activated receptor-gamma (PPARgamma) coactivator-1alpha (PGC-1alpha) is a major regulator of exercise-induced phenotypic adaptation and substrate utilization. We provide an overview of 1) the role of PGC-1alpha in exercise-mediated muscle adaptation and 2) the possible insulin-sensitizing role of PGC-1alpha. To these ends, the following questions are addressed. 1) How is PGC-1alpha regulated, 2) what adaptations are indeed dependent on PGC-1alpha action, 3) is PGC-1alpha altered in insulin resistance, and 4) are PGC-1alpha-knockout and -transgenic mice suitable models for examining therapeutic potential of this coactivator? In skeletal muscle, an orchestrated signaling network, including Ca(2+)-dependent pathways, reactive oxygen species (ROS), nitric oxide (NO), AMP-dependent protein kinase (AMPK), and p38 MAPK, is involved in the control of contractile protein expression, angiogenesis, mitochondrial biogenesis, and other adaptations. However, the p38gamma MAPK/PGC-1alpha regulatory axis has been confirmed to be required for exercise-induced angiogenesis and mitochondrial biogenesis but not for fiber type transformation. With respect to a potential insulin-sensitizing role of PGC-1alpha, human studies on type 2 diabetes suggest that PGC-1alpha and its target genes are only modestly downregulated (< or =34%). However, studies in PGC-1alpha-knockout or PGC-1alpha-transgenic mice have provided unexpected anomalies, which appear to suggest that PGC-1alpha does not have an insulin-sensitizing role. In contrast, a modest ( approximately 25%) upregulation of PGC-1alpha, within physiological limits, does improve mitochondrial biogenesis, fatty acid oxidation, and insulin sensitivity in healthy and insulin-resistant skeletal muscle. Taken altogether, there is substantial evidence that the p38gamma MAPK-PGC-1alpha regulatory axis is critical for exercise-induced metabolic adaptations in skeletal muscle, and strategies that upregulate PGC-1alpha, within physiological limits, have revealed its insulin-sensitizing effects.

High-intensity aerobic interval training increases fat and carbohydrate metabolic capacities in human skeletal muscle

High-intensity aerobic interval training (HIIT) is a compromise between time-consuming moderate-intensity training and sprint-interval training requiring all-out efforts. However, there are few data regarding the ability of HIIT to increase the capacities of fat and carbohydrate oxidation in skeletal muscle. Using untrained recreationally active individuals, we investigated skeletal muscle and whole-body metabolic adaptations that occurred following 6 weeks of HIIT (~1 h of 10 x 4 min intervals at ~90% of peak oxygen consumption (VO2 peak), separated by 2 min rest, 3 d.week-1). A VO2 peak test, a test to exhaustion (TE) at 90% of pre-training VO2 peak, and a 1 h cycle at 60% of pre-training VO2 peak were performed pre- and post-HIIT. Muscle biopsies were sampled during the TE at rest, after 5 min, and at exhaustion. Training power output increased by 21%, and VO2 peak increased by 9% following HIIT. Muscle adaptations at rest included the following: (i) increased cytochrome c oxidase IV content (18%) and maximal activities of the mitochondrial enzymes citrate synthase (26%), beta-hydroxyacyl-CoA dehydrogenase (29%), aspartate-amino transferase (26%), and pyruvate dehydrogenase (PDH; 21%); (ii) increased FAT/CD36, FABPpm, GLUT 4, and MCT 1 and 4 transport proteins (14%-30%); and (iii) increased glycogen content (59%). Major adaptations during exercise included the following: (i) reduced glycogenolysis, lactate accumulation, and substrate phosphorylation (0-5 min of TE); (ii) unchanged PDH activation (carbohydrate oxidation; 0-5 min of TE); (iii) ~2-fold greater time during the TE; and (iv) increased fat oxidation at 60% of pre-training VO2 peak. This study demonstrated that 18 h of repeated high-intensity exercise sessions over 6 weeks (3 d.week-1) is a powerful method to increase whole-body and skeletal muscle capacities to oxidize fat and carbohydrate in previously untrained individuals.