Co-overexpression of CD36 and FABPpm increases fatty acid transport additively, not synergistically, within muscle.

We aimed to determine the combined effects of overexpressing plasma membrane fatty acid binding protein (FABPpm) and fatty acid translocase (CD36) on skeletal muscle fatty acid transport to establish if these transport proteins function collaboratively. Electrotransfection with either FABPpm or CD36 increased their protein content at the plasma membrane (+75% and +64%), increased fatty acid transport rates by +24% for FABPpm and +62% for CD36, resulting in a calculated transport efficiency of ∼0.019 and ∼0.053 per unit protein change for FABPpm and CD36, respectively. We subsequently used these data to determine if increasing both proteins additively or synergistically increased fatty acid transport. Cotransfection of FABPpm and CD36 simultaneously increased protein content in whole muscle (FABPpm, +46%; CD36, +45%) and at the sarcolemma (FABPpm, +41%; CD36, +42%), as well as fatty acid transport rates (+50%). Since the relative effects of changing FABPpm and CD36 content had been independently determined, we were able to a predict a change in fatty acid transport based on the overexpression of plasmalemmal transporters in the cotransfection experiments. This prediction yielded an increase in fatty acid transport of +0.984 and +1.722 pmol/mg prot/15 s for FABPpm and CD36, respectively, for a total increase of +2.96 pmol/mg prot/15 s. This calculated determination was remarkably consistent with the measured change in transport, namely +2.89 pmol/mg prot/15 s. Altogether, these data indicate that increasing CD36 and FABPpm alters fatty acid transport rates additively, but not synergistically, suggesting an independent mechanism of action within muscle for each transporter. This conclusion was further supported by the observation that plasmalemmal CD36 and FABPpm did not coimmunoprecipitate.

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.

Regulation of the subcellular trafficking of CD36, a major determinant of cardiac fatty acid utilization.

Myocardial uptake of long-chain fatty acids largely occurs by facilitated diffusion, involving primarily the membrane-associated protein CD36. Other putative fatty acid transporters, such as FABPpm, FATP1 and FATP4, also play a role, but their quantitative contribution is much smaller or their involvement is rather permissive. Besides its sarcolemmal localization, CD36 is also present in intracellular compartments (endosomes). CD36 cycles between both pools via vesicle-mediated trafficking, and the relative distribution between endosomes versus sarcolemma determines the rate of cardiac fatty acid uptake. A net translocation of CD36 to the sarcolemma is induced by various stimuli, in particular hormones like insulin and myocyte contractions, so as to allow a proper coordination of the rate of fatty acid uptake with rapid fluctuations in myocardial energy needs. Furthermore, changes in cardiac fatty acid utilization that occur in both acute and chronic cardiac disease appear to be accompanied by concomitant changes in the sarcolemmal presence of CD36. Studies in various animal and cell models suggest that interventions aimed at modulating the sarcolemmal presence or functioning of CD36 hold promise as therapy to rectify aberrant rates of fatty acid uptake in order to fight cardiac metabolic remodeling and restore proper contractile function. In this review we discuss our current knowledge about the role of CD36 in cardiac fatty acid uptake and metabolism in health and disease with focus on the regulation of the subcellular trafficking of CD36 and its selective modulation as therapeutic approach for cardiac disease. This article is part of a Special Issue entitled: Heart Lipid Metabolism edited by G.D. Lopaschuk.

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.

Extremely rapid increase in fatty acid transport and intramyocellular lipid accumulation but markedly delayed insulin resistance after high fat feeding in rats.

AIMS/HYPOTHESIS: The mechanisms for diet-induced intramyocellular lipid accumulation and its association with insulin resistance remain contentious. In a detailed time-course study in rats, we examined whether a high-fat diet increased intramyocellular lipid accumulation via alterations in fatty acid translocase (FAT/CD36)-mediated fatty acid transport, selected enzymes and/or fatty acid oxidation, and whether intramyocellular lipid accretion coincided with the onset of insulin resistance. METHODS: We measured, daily (on days 1-7) and/or weekly (for 6 weeks), the diet-induced changes in circulating substrates, insulin, sarcolemmal substrate transporters and transport, selected enzymes, intramyocellular lipids, mitochondrial fatty acid oxidation and basal and insulin-stimulated sarcolemmal GLUT4 and glucose transport. We also examined whether upregulating fatty acid oxidation improved glucose transport in insulin-resistant muscles. Finally, in Cd36-knockout mice, we examined the role of FAT/CD36 in intramyocellular lipid accumulation, insulin sensitivity and diet-induced glucose intolerance. RESULTS: Within 2-3 days, diet-induced increases occurred in insulin, sarcolemmal FAT/CD36 (but not fatty acid binding protein [FABPpm] or fatty acid transporter [FATP]1 or 4), fatty acid transport and intramyocellular triacylglycerol, diacylglycerol and ceramide, independent of enzymatic changes or muscle fatty acid oxidation. Diet-induced increases in mitochondria and mitochondrial fatty acid oxidation and impairments in insulin-stimulated glucose transport and GLUT4 translocation occurred much later (≥21 days). FAT/CD36 ablation impaired insulin-stimulated fatty acid transport and lipid accumulation, improved insulin sensitivity and prevented diet-induced glucose intolerance. Increasing fatty acid oxidation in insulin-resistant muscles improved glucose transport. CONCLUSIONS/INTERPRETATIONS: High-fat feeding rapidly increases intramyocellular lipids (in 2-3 days) via insulin-mediated upregulation of sarcolemmal FAT/CD36 and fatty acid transport. The 16-19 day delay in the onset of insulin resistance suggests that additional mechanisms besides intramyocellular lipids contribute to this pathology.

Chronic muscle stimulation improves insulin sensitivity while increasing subcellular lipid droplets and reducing selected diacylglycerol and ceramide species in obese Zucker rats.

AIMS/HYPOTHESIS: Although insulin resistance has been associated with accumulations of specific intramuscular fatty acids and altered subcellular localisation of lipid droplets, these concepts remain controversial. Therefore, we aimed to identify specific intramuscular fatty acids and subcellular lipid localisations associated with improved insulin sensitivity following chronic muscle contraction. METHODS: In lean and insulin-resistant obese Zucker rats the tibialis anterior muscle was stimulated (6 h/day for 6 days). Thereafter, muscles were examined for insulin sensitivity, intramuscular lipid droplet localisation and triacylglycerol (TAG), diacylglycerol (DAG) and ceramide fatty acid composition. RESULTS: In lean and obese animals, regardless of muscle type, chronic muscle contraction improved muscle insulin sensitivity and increased intramuscular levels of total and most C14-C22 TAG fatty acids (p < 0.05). Therefore, accumulation in subcellular lipid droplet compartments reflected the oversupply of lipids within muscle. In contrast, improvements in insulin sensitivity induced by muscle contraction were associated with reductions in specific DAG and ceramide species that were not uniform in red and white muscle of obese rats. However, these reductions were insufficient to fully normalise insulin sensitivity, indicating that other mechanisms are involved. CONCLUSIONS/INTERPRETATION: Reductions in 18 C length DAG and ceramide species were the most consistent in red and white muscle and therefore may represent therapeutic targets for improving insulin sensitivity.

Calcium signaling recruits substrate transporters GLUT4 and CD36 to the sarcolemma without increasing cardiac substrate uptake.

Activation of AMP-activated protein kinase (AMPK) in cardiomyocytes induces translocation of glucose transporter GLUT4 and long-chain fatty acid (LCFA) transporter CD36 from endosomal stores to the sarcolemma to enhance glucose and LCFA uptake, respectively. Ca(2+)/calmodulin-activated kinase kinase-ß (CaMKKß) has been positioned directly upstream of AMPK. However, it is unknown whether acute increases in [Ca(2+)]i stimulate translocation of GLUT4 and CD36 and uptake of glucose and LCFA or whether Ca(2+) signaling converges with AMPK signaling to exert these actions. Therefore, we studied the interplay between Ca(2+) and AMPK signaling in regulation of cardiomyocyte substrate uptake. Exposure of primary cardiomyocytes to inhibitors or activators of Ca(2+) signaling affected neither AMPK-Thr(172) phosphorylation nor basal and AMPK-mediated glucose and LCFA uptake. Despite their lack of an effect on substrate uptake, Ca(2+) signaling activators induced GLUT4 and CD36 translocation. In contrast, AMPK activators stimulated GLUT4/CD36 translocation as well as glucose/LCFA uptake. When cardiomyocytes were cotreated with Ca(2+) signaling and AMPK activators, Ca(2+) signaling activators further enhanced AMPK-induced glucose/LCFA uptake. In conclusion, Ca(2+) signaling shows no involvement in AMPK-induced GLUT4/CD36 translocation and substrate uptake but elicits transporter translocation via a separate pathway requiring CaMKKß/CaMKs. Ca(2+)-induced transporter translocation by itself appears to be ineffective to increase substrate uptake but requires additional AMPK activation to effectuate transporter translocation into increased substrate uptake. Ca(2+)-induced transporter translocation might be crucial under excessive cardiac stress conditions that require supraphysiological energy demands. Alternatively, Ca(2+) signaling might prepare the heart for substrate uptake during physiological contraction by inducing transporter translocation.

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.

Overexpression of vesicle-associated membrane protein (VAMP) 3, but not VAMP2, protects glucose transporter (GLUT) 4 protein translocation in an in vitro model of cardiac insulin resistance.

Cardiac glucose utilization is regulated by reversible translocation of the glucose transporter GLUT4 from intracellular stores to the plasma membrane. During the onset of diet-induced insulin resistance, elevated lipid levels in the circulation interfere with insulin-stimulated GLUT4 translocation, leading to impaired glucose utilization. Recently, we identified vesicle-associated membrane protein (VAMP) 2 and 3 to be required for insulin- and contraction-stimulated GLUT4 translocation, respectively, in cardiomyocytes. Here, we investigated whether overexpression of VAMP2 and/or VAMP3 could protect insulin-stimulated GLUT4 translocation under conditions of insulin resistance. HL-1 atrial cardiomyocytes transiently overexpressing either VAMP2 or VAMP3 were cultured for 16 h with elevated concentrations of palmitate and insulin. Upon subsequent acute stimulation with insulin, we measured GLUT4 translocation, plasmalemmal presence of the fatty acid transporter CD36, and myocellular lipid accumulation. Overexpression of VAMP3, but not VAMP2, completely prevented lipid-induced inhibition of insulin-stimulated GLUT4 translocation. Furthermore, the plasmalemmal presence of CD36 and intracellular lipid levels remained normal in cells overexpressing VAMP3. However, insulin signaling was not retained, indicating an effect of VAMP3 overexpression downstream of PKB/Akt. Furthermore, we revealed that endogenous VAMP3 is bound by the contraction-activated protein kinase D (PKD), and contraction and VAMP3 overexpression protect insulin-stimulated GLUT4 translocation via a common mechanism. These observations indicate that PKD activates GLUT4 translocation via a VAMP3-dependent trafficking step, which pathway might be valuable to rescue constrained glucose utilization in the insulin-resistant heart.

Protein kinase D1 is essential for contraction-induced glucose uptake but is not involved in fatty acid uptake into cardiomyocytes.

Increased contraction enhances substrate uptake into cardiomyocytes via translocation of the glucose transporter GLUT4 and the long chain fatty acid (LCFA) transporter CD36 from intracellular stores to the sarcolemma. Additionally, contraction activates the signaling enzymes AMP-activated protein kinase (AMPK) and protein kinase D1 (PKD1). Although AMPK has been implicated in contraction-induced GLUT4 and CD36 translocation in cardiomyocytes, the precise role of PKD1 in these processes is not known. To study this, we triggered contractions in cardiomyocytes by electric field stimulation (EFS). First, the role of PKD1 in GLUT4 and CD36 translocation was defined. In PKD1 siRNA-treated cardiomyocytes as well as cardiomyocytes from PKD1 knock-out mice, EFS-induced translocation of GLUT4, but not CD36, was abolished. In AMPK siRNA-treated cardiomyocytes and cardiomyocytes from AMPKα2 knock-out mice, both GLUT4 and CD36 translocation were abrogated. Hence, unlike AMPK, PKD1 is selectively involved in glucose uptake. Second, we analyzed upstream factors in PKD1 activation. Cardiomyocyte contractions enhanced reactive oxygen species (ROS) production. Using ROS scavengers, we found that PKD1 signaling and glucose uptake are more sensitive to changes in intracellular ROS than AMPK signaling or LCFA uptake. Furthermore, silencing of death-activated protein kinase (DAPK) abrogated EFS-induced GLUT4 but not CD36 translocation. Finally, possible links between PKD1 and AMPK signaling were investigated. PKD1 silencing did not affect AMPK activation. Reciprocally, AMPK silencing did not alter PKD1 activation. In conclusion, we present a novel contraction-induced ROS-DAPK-PKD1 pathway in cardiomyocytes. This pathway is activated separately from AMPK and mediates GLUT4 translocation/glucose uptake, but not CD36 translocation/LCFA uptake.

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.

Ectopic lipid deposition and the metabolic profile of skeletal muscle in ovariectomized mice.

Disruptions of ovarian function in women are associated with increased risk of metabolic disease due to dysregulation of peripheral glucose homeostasis in skeletal muscle. Our previous evidence suggests that alterations in skeletal muscle lipid metabolism coupled with altered mitochondrial function may also develop. The objective of this study was to use an integrative metabolic approach to identify potential areas of dysfunction that develop in skeletal muscle from ovariectomized (OVX) female mice compared with age-matched ovary-intact adult female mice (sham). The OVX mice exhibited significant increases in body weight, visceral, and inguinal fat mass compared with sham mice. OVX mice also had significant increases in skeletal muscle intramyocellular lipids (IMCL) compared with the sham animals, which corresponded to significant increases in the protein content of the fatty acid transporters CD36/FAT and FABPpm. A targeted metabolic profiling approach identified significantly lower levels of specific acyl carnitine species and various amino acids in skeletal muscle from OVX mice compared with the sham animals, suggesting a potential dysfunction in lipid and amino acid metabolism, respectively. Basal and maximal mitochondrial oxygen consumption rates were significantly impaired in skeletal muscle fibers from OVX mice compared with sham animals. Collectively, these data indicate that loss of ovarian function results in increased IMCL storage that is coupled with alterations in mitochondrial function and changes in the skeletal muscle metabolic profile.

Monocarboxylate transporter 2 and stroke severity in a rodent model of sleep apnea.

Stroke is not only more prevalent but is also associated with more severe adverse functional outcomes among patients with sleep apnea. Monocarboxylate transporters (MCT) are important regulators of cellular bioenergetics, have been implicated in brain susceptibility to acute severe hypoxia (ASH), and could underlie the unfavorable prognosis of cerebrovascular accidents in sleep apnea patients. Rodents were exposed to either intermittent hypoxia (IH) during sleep, a characteristic feature of sleep apnea, or to sustained hypoxia (SH), and expression of MCT1 and MCT2 was assessed. In addition, the functional recovery to middle cerebral artery occlusion (MCAO) in rats and hMCT2 transgenic mice and of hippocampal slices subjected to ASH was assessed, as well as the effects of MCT blocker and MCT2 antisense oligonucleotides and siRNAs. IH, but not SH, induced significant reductions in MCT2 expression over time at both the mRNA and protein levels and in the functional recovery of hippocampal slices subjected to ASH. Similarly, MCAO-induced infarcts were significantly greater in IH-exposed rats and mice, and overexpression of hMCT2 in mice markedly attenuated the adverse effects of IH. Exogenous pyruvate treatment reduced infarct volumes in normoxic rats but not in IH-exposed rats. Administration of the MCT2 blocker 4CN, but not the MCT1 antagonist p-chloromercuribenzene sulfonate, increased infarct size. Thus, prolonged exposures to IH mimicking sleep apnea are associated with increased CNS vulnerability to ischemia that is mediated, at least in part, by concomitant decreases in the expression and function of MCT2. Efforts to develop agonists of MCT2 should provide opportunities to ameliorate the overall outcome of stroke.

Acute endurance exercise increases plasma membrane fatty acid transport proteins in rat and human skeletal muscle.

Fatty acid transport proteins are present on the plasma membrane and are involved in the uptake of long-chain fatty acids into skeletal muscle. The present study determined whether acute endurance exercise increased the plasma membrane content of fatty acid transport proteins in rat and human skeletal muscle and whether the increase was accompanied by an increase in long-chain fatty acid transport in rat skeletal muscle. Sixteen subjects cycled for 120 min at ∼60 ± 2% Vo(2) peak. Two skeletal muscle biopsies were taken at rest and again following cycling. In a parallel study, eight Sprague-Dawley rats ran for 120 min at 20 m/min, whereas eight rats acted as nonrunning controls. Giant sarcolemmal vesicles were prepared, and protein content of FAT/CD36 and FABPpm was measured in human and rat vesicles and whole muscle homogenate. Palmitate uptake was measured in the rat vesicles. In human muscle, plasma membrane FAT/CD36 and FABPpm protein contents increased 75 and 20%, respectively, following 120 min of exercise. In rat muscle, plasma membrane FAT/CD36 and FABPpm increased 20 and 30%, respectively, and correlated with a 30% increase in palmitate transport following 120 min of running. These data suggest that the translocation of FAT/CD36 and FABPpm to the plasma membrane in rat skeletal muscle is related to the increase in fatty acid transport and oxidation that occurs with endurance running. This study is also the first to demonstrate that endurance cycling induces an increase in plasma membrane FAT/CD36 and FABPpm content in human skeletal muscle, which is predicted to increase fatty acid transport.

Consumption of a high-fat diet rapidly exacerbates the development of fatty liver disease that occurs with chronically elevated glucocorticoids.

Chronically elevated glucocorticoids (GCs) and a high-fat diet (HFD) independently induce insulin resistance, abdominal obesity, and nonalcoholic fatty liver disease (NAFLD). GCs have been linked to increased food intake, particularly energy-dense "comfort" foods. Thus we examined the synergistic actions of GCs and HFD on hepatic disease development in a new rodent model of chronically elevated GCs. Six-week-old male Sprague-Dawley rats received exogenous GCs, via subcutaneous implantation of four 100-mg corticosterone (Cort) pellets, to elevate basal GC levels for 16 days (n = 8-10 per group). Another subset of animals received wax pellets (placebo) to serve as controls. Animals from each group were randomly assigned to receive a 60% HFD or a standard high-carbohydrate (13% fat and 60% carbohydrate) diet. Cort + HFD resulted in central obesity, despite a relative weight loss, a 4-fold increase in hepatic lipid content, hepatic fibrosis, and a 2.8-fold increase in plasma alanine aminotransferase levels compared with placebo + chow controls. Hepatic injury developed independent of inflammation, as plasma haptoglobin levels were reduced with Cort treatment. Insulin resistance and hepatic steatosis occurred with Cort alone; these outcomes were further exacerbated by the HFD in the presence of elevated Cort. In addition to fatty liver, the Cort + HFD group also developed severe insulin resistance, hyperinsulinemia, hyperglycemia, and hypertriglyceridemia, which were not evident with HFD or Cort alone. Thus a HFD dramatically exacerbates the development of NAFLD and characteristics of the metabolic syndrome in conditions of chronically elevated Cort.

Clenbuterol, a ß2-adrenergic agonist, reciprocally alters PGC-1 alpha and RIP140 and reduces fatty acid and pyruvate oxidation in rat skeletal muscle.

Clenbuterol, a ß2-adrenergic agonist, reduces mitochondrial content and enzyme activities in skeletal muscle, but the mechanism involved has yet to be identified. We examined whether clenbuterol-induced changes in the muscles' metabolic profile and the intrinsic capacity of mitochondria to oxidize substrates are associated with reductions in the nuclear receptor coactivator PGC-1 alpha and/or an increase in the nuclear corepressor RIP140. In rats, clenbuterol was provided in the drinking water (30 mg/l). In 3 wk, this increased body (8%) and muscle weights (12-17%). In red (R) and white (W) muscles, clenbuterol induced reductions in mitochondrial content (citrate synthase: R, 27%; W, 52%; cytochrome-c oxidase: R, 24%; W, 34%), proteins involved in fatty acid transport (fatty acid translocase/CD36: R, 36%; W, 35%) and oxidation [ß-hydroxyacyl CoA dehydrogenase (ß-HAD): R, 33%; W, 62%], glucose transport (GLUT4: R, 8%; W, 13%), lactate transport monocarboxylate transporter (MCT1: R, 61%; W, 37%), and pyruvate oxidation (PDHE1α, R, 18%; W, 12%). Concurrently, only red muscle lactate dehydrogenase activity (25%) and MCT4 (31%) were increased. Palmitate oxidation was reduced in subsarcolemmal (SS) (R, 30%; W, 52%) and intermyofibrillar (IMF) mitochondria (R, 17%; W, 44%) along with reductions in ß-HAD activity (SS: R, 17%; W, 51%; IMF: R, 20%; W, 57%). Pyruvate oxidation was only reduced in SS mitochondria (R, 20%; W, 28%), but this was not attributable solely to PDHE1α, which was reduced in both SS (R, 21%; W, 20%) and IMF mitochondria (R, 15%; W, 43%). These extensive metabolic changes induced by clenbuterol were associated with reductions in PGC-1α (R, 37%; W, 32%) and increases in RIP140 (R, 23%; W, 21%). This is the first evidence that clenbuterol appears to exert its metabolic effects via simultaneous and reciprocal changes in the nuclear receptor coactivator PGC-1α and the nuclear corepressor RIP140.

A dual mechanism of action for skeletal muscle FAT/CD36 during exercise.

Carnitine palmitoyltransferase I has been viewed historically as the sole regulator of fatty acid oxidation. However, we have identified fatty acid translocase/CD36 as an additional control point. Specifically, fatty acid translocase/CD36 seems to have a novel dual mechanism of action with regard to fatty acid oxidation during exercise, influencing transport of lipids across the sarcolemmal membrane and into the mitochondria.

FAT/CD36 is located on the outer mitochondrial membrane, upstream of long-chain acyl-CoA synthetase, and regulates palmitate oxidation.

FAT/CD36 (fatty acid translocase/Cluster of Differentiation 36), a plasma membrane fatty-acid transport protein, has been found on mitochondrial membranes; however, it remains unclear where FAT/CD36 resides on this organelle or its functional role within mitochondria. In the present study, we demonstrate, using several different approaches, that in skeletal muscle FAT/CD36 resides on the OMM (outer mitochondrial membrane). To determine the functional role of mitochondrial FAT/CD36 in this tissue, we determined oxygen consumption rates in permeabilized muscle fibres in WT (wild-type) and FAT/CD36-KO (knockout) mice using a variety of substrates. Despite comparable muscle mitochondrial content, as assessed by unaltered mtDNA (mitochondrial DNA), citrate synthase, ß-hydroxyacyl-CoA dehydrogenase, cytochrome c oxidase complex IV and respiratory capacities [maximal OXPHOS (oxidative phosphorylation) respiration] in WT and KO mice, palmitate-supported respiration was 34% lower in KO animals. In contrast, palmitoyl-CoA-supported respiration was unchanged. These results indicate that FAT/CD36 is key for palmitate-supported respiration. Therefore we propose a working model of mitochondrial fatty-acid transport, in which FAT/CD36 is positioned on the OMM, upstream of long-chain acyl-CoA synthetase, thereby contributing to the regulation of mitochondrial fatty-acid transport. We further support this model by providing evidence that FAT/CD36 is not located in mitochondrial contact sites, and therefore does not directly interact with carnitine palmitoyltransferase-I as original proposed.

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

We aimed to determine whether an increased rate of long-chain fatty acid (LCFA) transport and/or a reduction in mitochondrial oxidation contributes to lipid deposition in hearts, as lipid accumulation within cardiac muscle has been associated with heart failure. In hearts of lean and obese Zucker rats we examined: (a) triacylglycerol (TAG) and mitochondrial content and distribution using transmission electron microscopy (TEM), (b) LCFA oxidation in cardiac myocytes, and in isolated subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondria, and (c) rates of LCFA transport into cardiac vesicles. Compared to lean rats, in obese Zucker rats, lipid droplet size was similar but there were more (P < 0.05) droplets, and TAG esterification rates and contents were markedly increased. TEM analyses and biochemical determinations showed that SS and IMF mitochondria in obese animals did not appear to be different in their appearance, area, density and number, nor in citrate synthase, ß-hydroxy-acyl-CoA dehydrogenase and carnitine palmitoyl-transferase-I enzymatic activities, electron transport chain proteins, nor in their rates of LCFA oxidation either in cardiac myocytes or in isolated SS and IMF mitochondria (P > 0.05). In contrast, sarcolemmal plasma membrane fatty acid binding protein (FABPpm) and fatty acid translocase (FAT/CD36) protein and palmitate transport rates into cardiac vesicles were increased (P < 0.05; +50%) in obese animals. Collectively these data indicate that mitochondrial dysfunction in LCFA oxidation is not responsible for lipid accumulation in obese Zucker rat hearts. Rather, increased sarcolemmal LCFA transport proteins and rates of LCFA transport result in a greater number of lipid droplets within cardiac muscle.

Recovered insulin response by 2 weeks of leptin administration in high-fat fed rats is associated with restored AS160 activation and decreased reactive lipid accumulation.

Leptin is an adipokine that increases fatty acid (FA) oxidation, decreases intramuscular lipid stores, and improves insulin response in skeletal muscle. In an attempt to elucidate the underlying mechanisms by which these metabolic changes occur, we administered leptin (Lep) or saline (Sal) by miniosmotic pumps to rats during the final 2 wk of a 6-wk low-fat (LF) or high-fat (HF) diet. Insulin-stimulated glucose transport was impaired by the HF diet (HF-Sal) but was restored with leptin administration (HF-Lep). This improvement was associated with restored phosphorylation of Akt and AS160 and decreased in reactive lipid species (ceramide, diacylglycerol), known inhibitors of the insulin-signaling cascade. Total muscle citrate synthase (CS) activity was increased by both leptin and HF diet, but was not additive. Leptin increased subsarcolemmal (SS) and intramyofibrillar (IMF) mitochondria CS activity. Total muscle, sarcolemmal, and mitochondrial (SS and IMF) FA transporter (FAT/CD36) protein content was significantly increased with the HF diet, but not altered by leptin. Therefore, the decrease in reactive lipid stores and subsequent improvement in insulin response, secondary to leptin administration in rats fed a HF diet was not due to a decrease in FA transport protein content or altered cellular distribution.