Laelie A. Snook

Greater Transport Efficiencies of the Membrane Fatty Acid Transporters FAT/CD36 and FATP4 Compared with FABPpm and FATP1 and Differential Effects on Fatty Acid Esterification and Oxidation in Rat Skeletal Muscle

In selected mammalian tissues, long chain fatty acid transporters (FABPpm, FAT/CD36, FATP1, and FATP4) are co-expressed. There is controversy as to whether they all function as membrane-bound transporters and whether they channel fatty acids to oxidation and/or esterification. Among skeletal muscles, the protein expression of FABPpm, FAT/CD36, and FATP4, but not FATP1, correlated highly with the capacities for oxidative metabolism (r ≥ 0.94), fatty acid oxidation (r ≥ 0.88), and triacylglycerol esterification (r ≥ 0.87). We overexpressed independently FABPpm, FAT/CD36, FATP1, and FATP4, within a normal physiologic range, in rat skeletal muscle, to determine the effects on fatty acid transport and metabolism. Independent overexpression of each fatty acid transporter occurred without altering either the expression or plasmalemmal content of other fatty acid transporters. All transporters increased fatty acid transport, but FAT/CD36 and FATP4 were 2.3- and 1.7-fold more effective than FABPpm and FATP1, respectively. Fatty acid transporters failed to alter the rates of fatty acid esterification into triacylglycerols. In contrast, all transporters increased the rates of long chain fatty acid oxidation, but the effects of FABPpm and FAT/CD36 were 3-fold greater than for FATP1 and FATP4. Thus, fatty acid transporters exhibit different capacities for fatty acid transport and metabolism. In vivo, FAT/CD36 and FATP4 are the most effective fatty acid transporters, whereas FABPpm and FAT/CD36 are key for stimulating fatty acid oxidation.

Adiponectin resistance precedes the accumulation of skeletal muscle lipids and insulin resistance in high-fat-fed rats

High-fat (HF) diets can induce insulin resistance (IR) by altering skeletal muscle lipid metabolism. An imbalance between fatty acid (FA) uptake and oxidation results in intramuscular lipid accumulation, which can impair the insulin-signaling cascade. Adiponectin (Ad) is an insulin-sensitizing adipokine known to stimulate skeletal muscle FA oxidation and reduce lipid accumulation. Evidence of Ad resistance has been shown in obesity and following chronic HF feeding and may contribute to lipid accumulation observed in these conditions. Whether Ad resistance precedes and is associated with the development of IR is unknown. We conducted a time course HF feeding trial for 3 days, 2 wk, or 4 wk to determine the onset of Ad resistance and identify the ensuing changes in lipid metabolism and insulin signaling leading to IR in skeletal muscle. Ad stimulated FA oxidation (+28%, P < or = 0.05) and acetyl-CoA carboxylase phosphorylation (+34%, P < or = 0.05) in control animals but failed to do so in any HF-fed group (i.e., as early as 3 days). By 2 wk, plasma membrane FA transporters and intramuscular diacylglycerol (DAG) and ceramide were increased, and insulin-stimulated phosphorylation of both protein kinase B and protein kinase B substrate 160 was blunted compared with control animals. After 4 wk of HF feeding, maximal insulin-stimulated glucose transport was impaired compared with control. Taken together, our results demonstrate that an early loss of Ads stimulatory effect on FA oxidation precedes an increase in plasmalemmal FA transporters and the accumulation of intramuscular DAG and ceramide, blunted insulin signaling, and ultimately impaired maximal insulin-stimulated glucose transport in skeletal muscle induced by HF diets.

In obese rat muscle transport of palmitate is increased and is channeled to triacylglycerol storage despite an increase in mitochondrial palmitate oxidation

Intramuscular triacylglycerol (IMTG) accumulation in obesity has been attributed to increased fatty acid transport and/or to alterations in mitochondrial fatty acid oxidation. Alternatively, an imbalance in these two processes may channel fatty acids into storage. Therefore, in red and white muscles of lean and obese Zucker rats, we examined whether the increase in IMTG accumulation was attributable to an increased rate of fatty acid transport rather than alterations in subsarcolemmal (SS) or intermyofibrillar (IMF) mitochondrial fatty acid oxidation. In obese animals selected parameters were upregulated, including palmitate transport (red: +100%; white: +51%), plasmalemmal FAT/CD36 (red: +116%; white: +115%; not plasmalemmal FABPpm, FATP1, or FATP4), IMTG concentrations (red: approximately 2-fold; white: approximately 4-fold), and mitochondrial content (red +30%). Selected mitochondrial parameters were also greater in obese animals, namely, palmitate oxidation (SS red: +91%; SS white: +26%; not IMF mitochondria), FAT/CD36 (SS: +65%; IMF: +65%), citrate synthase (SS: +19%), and beta-hydroxyacyl-CoA dehydrogenase activities (SS: +20%); carnitine palmitoyltransferase-I activity did not differ. A comparison of lean and obese rat muscles revealed that the rate of change in IMTG concentration was eightfold greater than that of fatty acid oxidation (SS mitochondria), when both parameters were expressed relative to fatty transport. Thus fatty acid transport, esterification, and oxidation (SS mitochondria) are upregulated in muscles of obese Zucker rats, with these effects being most pronounced in red muscle. The additional fatty acid taken up is channeled primarily to esterification, suggesting that upregulation in fatty acid transport as opposed to altered fatty acid oxidation is the major determinant of intramuscular lipid accumulation.

Exercise training increases sarcolemmal and mitochondrial fatty acid transport proteins in human skeletal muscle.

Fatty acid oxidation is highly regulated in skeletal muscle and involves several sites of regulation, including the transport of fatty acids across both the plasma and mitochondrial membranes. Transport across these membranes is recognized to be primarily protein mediated, limited by the abundance of fatty acid transport proteins on the respective membranes. In recent years, evidence has shown that fatty acid transport proteins move in response to acute and chronic perturbations; however, in human skeletal muscle the localization of fatty acid transport proteins in response to training has not been examined. Therefore, we determined whether high-intensity interval training (HIIT) increased total skeletal muscle, sarcolemmal, and mitochondrial membrane fatty acid transport protein contents. Ten untrained females (22 +/- 1 yr, 65 +/- 2 kg; .VO(2peak): 2.8 +/- 0.1 l/min) completed 6 wk of HIIT, and biopsies from the vastus lateralis muscle were taken before training, and following 2 and 6 wk of HIIT. Training significantly increased maximal oxygen uptake at 2 and 6 wk (3.1 +/- 0.1, 3.3 +/- 0.1 l/min). Training for 6 wk increased FAT/CD36 at the whole muscle (10%) and mitochondrial levels (51%) without alterations in sarcolemmal content. Whole muscle plasma membrane fatty acid binding protein (FABPpm) also increased (48%) after 6 wk of training, but in contrast to FAT/CD36, sarcolemmal FABPpm increased (23%), whereas mitochondrial FABPpm was unaltered. The changes on sarcolemmal and mitochondrial membranes occurred rapidly, since differences (< or =2 wk) were not observed between 2 and 6 wk. This is the first study to demonstrate that exercise training increases fatty acid transport protein content in whole muscle (FAT/CD36 and FABPpm) and sarcolemmal (FABPpm) and mitochondrial (FAT/CD36) membranes in human skeletal muscle of females. These results suggest that increases in skeletal muscle fatty acid oxidation following training are related in part to changes in fatty acid transport protein content and localization.

Additive effects of insulin and muscle contraction on fatty acid transport and fatty acid transporters, FAT/CD36, FABPpm, FATP1, 4 and 6

Insulin and muscle contraction increase fatty acid transport into muscle by inducing the translocation of FAT/CD36. We examined (a) whether these effects are additive, and (b) whether other fatty acid transporters (FABPpm, FATP1, FATP4, and FATP6) are also induced to translocate. Insulin and muscle contraction increased glucose transport and plasmalemmal GLUT4 independently and additively (positive control). Palmitate transport was also stimulated independently and additively by insulin and by muscle contraction. Insulin and muscle contraction increased plasmalemmal FAT/CD36, FABPpm, FATP1, and FATP4, but not FATP6. Only FAT/CD36 and FATP1 were stimulated in an additive manner by insulin and by muscle contraction.

In Vivo, Fatty Acid Translocase (CD36) Critically Regulates Skeletal Muscle Fuel Selection, Exercise Performance, and Training-induced Adaptation of Fatty Acid Oxidation

Background: CD36-mediated lipid transport may regulate muscle fuel selection and adaptation. Results: CD36 ablation impaired fatty acid oxidation and prevented its exercise training-induced up-regulation. Without altering mitochondrial content, CD36 overexpression mimicked exercise training effects on fatty acid oxidation. Conclusion: CD36 contributes to regulating fatty acid oxidation and adaptation in a mitochondrion-independent manner. Significance: This work identified another mechanism regulating muscle 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, VO2max, and mitochondrial content and enzymes did not differ. In acutely exercised (78% VO2max) 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 β-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.

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

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.

Compensatory increases in nuclear PGC1α protein are primarily associated with subsarcolemmal mitochondrial adaptations in ZDF rats

OBJECTIVE We examined in insulin-resistant muscle if, in contrast to long-standing dogma, mitochondrial fatty acid oxidation is increased and whether this is attributed to an increased nuclear content of peroxisome proliferator–activated receptor (PPAR) γ coactivator (PGC) 1α and the adaptations of specific mitochondrial subpopulations. RESEARCH DESIGN AND METHODS Skeletal muscles from male control and Zucker diabetic fatty (ZDF) rats were used to determine 1) intramuscular lipid distribution, 2) subsarcolemmal and intermyofibrillar mitochondrial morphology, 3) rates of palmitate oxidation in subsarcolemmal and intermyofibrillar mitochondria, and 4) the subcellular localization of PGC1α. Electotransfection of PGC1α cDNA into lean animals tested the notion that increased nuclear PGC1α preferentially targeted subsarcolemmal mitochondria. RESULTS Transmission electron microscope analysis revealed that in ZDF animals the number (+50%), width (+69%), and density (+57%) of subsarcolemmal mitochondria were increased (P < 0.05). In contrast, intermyofibrillar mitochondria remained largely unchanged. Rates of palmitate oxidation were ∼40% higher (P < 0.05) in ZDF subsarcolemmal and intermyofibrillar mitochondria, potentially as a result of the increased PPAR-targeted proteins, carnitine palmitoyltransferase-I, and fatty acid translocase (FAT)/CD36. PGC1α mRNA and total protein were not altered in ZDF animals; however, a greater (∼70%; P < 0.05) amount of PGC1α was located in nuclei. Overexpression of PGC1α only increased subsarcolemmal mitochondrial oxidation rates. CONCLUSIONS In ZDF animals, intramuscular lipids accumulate in the intermyofibrillar region (increased size and number), and this is primarily associated with increased oxidative capacity in subsarcolemmal mitochondria (number, size, density, and oxidation rates). These changes may result from an increased nuclear content of PGC1α, as under basal conditions, overexpression of PGC1α appears to target subsarcolemmal mitochondria.

Resveratrol supplementation improves white adipose tissue function in a depot-specific manner in Zucker diabetic fatty rats

Resveratrol (RSV) is a polyphenolic compound suggested to have anti-diabetic properties. Surprisingly, little is known regarding the effects of RSV supplementation on adipose tissue (AT) metabolism in vivo. The purpose of this study was to assess the effects of RSV on mitochondrial content and respiration, glyceroneogenesis (GNG), and adiponectin secretion in adipose tissue from Zucker diabetic fatty (ZDF) rats. Five-week-old ZDF rats were fed a chow diet with (ZDF RSV) or without (ZDF chow) RSV (200 mg/kg body wt) for 6 wk. Changes in adipose tissue metabolism were assessed in subcutaneous (scAT) and intra-abdominal [retroperitoneal (rpWAT), epididymal (eWAT)] adipose tissue depots. ZDF RSV rats showed lower fasting glucose and higher circulating adiponectin, as well as lower glucose area under the curve during intraperitoneal glucose and insulin tolerance tests than ZDF chow. [¹⁴C]pyruvate incorporation into triglycerides and adiponectin secretion were higher in scAT from ZDF RSV rats, concurrent with increases in adipose tissue triglyceride lipase (ATGL), hormone-sensitive lipase (HSL), and the phosphorylation of pyruvate dehydrogenase-E1α (PDH) (Ser293) protein content in this depot. Moreover, uncoupled mitochondrial respiration and complex I and II-supported respiration were increased in both scAT and rpWAT, which correlated with increases in cytochrome c oxidase subunit IV (COX4) protein content. In vitro treatment of scAT with RSV (50 μmol/l; 24 h) induced pyruvate dehydrogenase kinase 4 (PDK4) and peroxisome proliferator-activated receptor (PPAR)-γ coactivator-1α (PGC-1α) mRNA expression. Collectively, these data demonstrate that RSV can induce adipose tissue mitochondrial biogenesis in parallel with increases in GNG and adiponectin secretion.

In obese Zucker rats, lipids accumulate in the heart despite normal mitochondrial content, morphology and long‐chain fatty acid oxidation

The storage of fat within the heart muscle has been associated with reductions in force production, which has implications for the ability of the heart to adequately pump blood. With the assistance of membrane proteins known as transport proteins, fats from the blood can be moved into heart muscle cells, where they can either be stored or used for generating energy (within a structure called mitochondria) to pump blood. We provide evidence that in obese animals more fat accumulates within the heart as a result of their increased transport across the membranes of heart cells, not due to reductions in mitochondrial number or function. The knowledge of why fat accumulates in the heart may provide insight into novel treatments/therapies, and the current study suggests therapies focused on limiting the entry of fats into the heart may restore the ability of the heart to pump blood.