From fat to FAT (CD36/SR-B2):Understanding the regulation of cellular fatty acid uptake

The molecular mechanisms underlying the cellular uptake of long-chain fatty acids and the regulation of this process have been elucidated in appreciable detail in the last decades. Two main players in this field, each discovered in the early 1990s, are (i) a membrane-associated protein first identified in adipose ('fat') tissue and referred to as putative fatty acid translocase (FAT)/CD36 (now officially designated as SR-B2) which facilitates the transport of fatty acids across the plasma membrane, and (ii) the family of transcription factors designated peroxisome proliferator-activated receptors (PPAR alpha, PPAR gamma, and PPAR(beta/delta) for which fatty acids and fatty acid metabolites are the preferred ligand. CD36/SR-B2 is the predominant membrane protein involved in fatty acid uptake into intestinal enterocytes, adipocytes and cardiac and skeletal myocytes. The rate of cellular fatty acid uptake is regulated by the subcellular vesicular recycling of CD36/SR-B2 from endosomes to the plasma membrane. Fatty acid-induced activation of PPARs results in the upregulation of the expression of genes encoding various proteins and enzymes involved in cellular fatty acid utilization. Both CD36/SR-B2 and the PPARs have been implicated in the derangements in fatty acid and lipid metabolism occurring with the development of pathophysiological conditions, such as high fat diet-induced insulin resistance and diabetic cardiomyopathy, and have been suggested as targets for metabolic intervention. In this brief review we discuss the discovery and current understanding of both CD36/SR-B2 and the PPARs in metabolic homeostasis. (C) 2016 Elsevier B.V. and Societe Francaise de Biochimie et Biologie Moleculaire (SFBBM). All rights reserved

Caffeine-stimulated fatty acid oxidation is blunted in CD36 null mice

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

Increased levels of peroxisome proliferator-activated receptor gamma, coactivator 1 alpha (PGC-1 alpha) improve lipid utilisation, insulin signalling and glucose transport in skeletal muscle of lean and insulin-resistant obese Zucker rats

Aims/hypothesis Reductions in peroxisome proliferator-activated receptor gamma, coactivator 1 alpha (PGC-1 alpha) levels have been associated with the skeletal muscle insulin resistance. However, in vivo, the therapeutic potential of PGC-1 alpha has met with failure, as supra-physiological overexpression of PGC-1 alpha induced insulin resistance, due to fatty acid translocase (FAT)-mediated lipid accumulation. Based on physiological and metabolic considerations, we hypothesised that a modest increase in PGC-1 alpha levels would limit FAT upregulation and improve lipid metabolism and insulin sensitivity, although these effects may differ in lean and insulin-resistant muscle. Methods Pgc-1 alpha was transfected into lean and obese Zucker rat muscles. Two weeks later we examined mitochondrial biogenesis, intramuscular lipids (triacylglycerol, diacylglycerol, ceramide), GLUT4 and FAT levels, insulin-stimulated glucose transport and signalling protein phosphorylation (thymoma viral proto-oncogene 2 [Akt2], Akt substrate of 160 kDa [AS160]), and fatty acid oxidation in subsarcolemmal and intermyofibrillar mitochondria. Results Electrotransfection yielded physiologically relevant increases in Pgc-1 alpha (also known as Ppargc1a) mRNA and protein (similar to 25%) in lean and obese muscle. This induced mitochondrial biogenesis, and increased FAT and GLUT4 levels, insulin-stimulated glucose transport, and Akt2 and AS160 phosphorylation in lean and obese animals, while bioactive intramuscular lipids were only reduced in obese muscle. Concurrently, PGC-1 alpha increased palmitate oxidation in subsarcolemmal, but not in intermyofibrillar mitochondria, in both groups. In obese compared with lean animals, the PGC-1 alpha-induced improvement in insulin-stimulated glucose transport was smaller, but intramuscular lipid reduction was greater. Conclusions/interpretations Increases in PGC-1 alpha levels, similar to those that can be induced by physiological stimuli, altered intramuscular lipids and improved fatty acid oxidation, insulin signalling and insulin-stimulated glucose transport, albeit to different extents in lean and insulin-resistant muscle. These positive effects are probably attributable to limiting the PGC-1 alpha-induced increase in FAT, thereby preventing bioactive lipid accumulation as has occurred in transgenic PGC-1 alpha animals

Munc18c provides stimulus-selective regulation of GLUT4 but not fatty acid transporter trafficking in skeletal muscle

Insulin-, and contraction-induced GLUT4 and fatty acid (FA) transporter translocation may share common trafficking mechanisms. Our objective was to examine the effects of partial Munc18c ablation on muscle glucose and FA transport, FA oxidation, GLUT4 and FA transporter (FAT/CD36, FAB-Ppm, FATP1, FATP4) trafficking to the sarcolemma, and FAT/CD36 to mitochondria. In Munc18c(-/+) mice, insulin-stimulated glucose transport and GLUT4 sarcolemmal appearance were impaired, but were unaffected by contraction. Insulin- and contraction-stimulated FA transport, sarcolemmal FA transporter appearance, and contraction-mediated mitochondrial FAT/CD36 were increased normally in Munc18c(-/+) mice. Hence, Munc18c provides stimulus-specific regulation of GLUT4 trafficking, but not FA transporter trafficking

Requirement for distinct vesicle-associated membrane proteins in insulin- and AMP-activated protein kinase (AMPK)-induced translocation of GLUT4 and CD36 in cultured cardiomyocytes

Upon stimulation of insulin signalling or contraction-induced AMP-activated protein kinase (AMPK) activation, the glucose transporter GLUT4 and the long-chain fatty acid (LCFA) transporter CD36 similarly translocate from intracellular compartments to the plasma membrane of cardiomyocytes to increase uptake of glucose and LCFA, respectively. This similarity in regulation of GLUT4 traffic and CD36 traffic suggests that the same families of trafficking proteins, including vesicle-associated membrane proteins (VAMPs), are involved in both processes. While several VAMPs have been implicated in GLUT4 traffic, nothing is known about the putative function of VAMPs in CD36 traffic. Therefore, we compared the involvement of the myocardially produced VAMP isoforms in insulin- or contraction-induced GLUT4 and CD36 translocation. Five VAMP isoforms were silenced in HL-1 cardiomyocytes. The cells were treated with insulin or the contraction-like AMPK activator oligomycin or were electrically stimulated to contract. Subsequently, GLUT4 and CD36 translocation as well as substrate uptake were measured. Three VAMPs were demonstrated to be necessary for both GLUT4 and CD36 translocation, either specifically in insulin-treated cells (VAMP2, VAMP5) or in oligomycin/contraction-treated cells (VAMP3). In addition, there are VAMPs specifically involved in either GLUT4 traffic (VAMP7 mediates basal GLUT4 retention) or CD36 traffic (VAMP4 mediates insulin- and oligomycin/contraction-induced CD36 translocation). The involvement of distinct VAMP isoforms in both GLUT4 and CD36 translocation indicates that CD36 translocation, just like GLUT4 translocation, is a vesicle-mediated process dependent on soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex formation. The ability of other VAMPs to discriminate between GLUT4 and CD36 translocation allows the notion that myocardial substrate preference can be modulated by these VAMPs

The interaction between AMPK beta 2 and the PP1-targeting subunit R6 is dynamically regulated by intracellular glycogen content

11 páginas, 7 figuras.AMP-activated protein kinase (AMPK) is a metabolic stress-sensing kinase. We previously showed that glucose deprivation induces autophosphorylation of AMPKβ at threonine-148 (Thr-148), which prevents the binding of AMPK to glycogen. Furthermore, in MIN6 cells, AMPKβ1 binds to R6 (PPP1R3D), a glycogen-targeting subunit of protein phosphatase 1 (PP1), thereby regulating the glucose-induced inactivation of AMPK. Here, we further investigated the interaction of R6 with AMPKβ and the possible dependency on Thr-148 phosphorylation status. Yeast two-hybrid analyses and co-immunoprecipitation of the overexpressed proteins in HEK293T cells revealed that both AMPKβ1 and β2 wild-type (WT) isoforms bind to R6. The AMPKβ/R6 interaction was stronger with the muscle-specific β2-WT and required association with the substrate-binding motif of R6. When HEK293T cells or C2C12 myotubes were cultured in high-glucose medium, AMPKβ2-WT and R6 weakly interacted. In contrast, glycogen depletion significantly enhanced this protein interaction. Mutation of AMPKβ2 Thr-148 prevented the interaction with R6 irrespective of the intracellular glycogen content. Treatment with the AMPK activator oligomycin enhanced AMPKβ2/R6 interaction in conjunction with increased Thr-148 phosphorylation in cells grown in low glucose medium. These data are in accordance with R6 binding directly to AMPKβ2 when both proteins detach from the diminishing glycogen particle, which is simultaneous to increased AMPKβ2 Thr-148 autophosphorylation. Such model points to a possible control of AMPK by PP1-R6 upon glycogen depletion in muscle.DN is recipient of a VIDI-Innovational Research Grant from the Netherlands Organization of Scientific Research (NWO-ALW Grant no. 864.10.007). This work has further been supported by grants from the Spanish Ministry of Education and Science SAF2014-54604-C3-1-R and a grant from Generalitat Valenciana (PrometeoII/2014/029) to PS.Peer reviewe

Palmitate-Induced Vacuolar-Type H(+)-ATPase Inhibition Feeds Forward Into Insulin Resistance and Contractile Dysfunction

Dietary fat overconsumption leads to myocardial lipid accumulation through mechanisms that are incompletely resolved. Previously, we identified increased translocation of the fatty acid transporter CD36 from its endosomal storage compartment to the sarcolemma as the primary mechanism of excessive myocellular lipid import. Here, we show that increased CD36 translocation is caused by alkalinization of endosomes resulting from inhibition of proton pumping activity of vacuolar-type H+-ATPase (v-ATPase). Endosomal alkalinization was observed in hearts from rats fed a lard-based high-fat diet and in rodent and human cardiomyocytes upon palmitate overexposure, and appeared as an early lipid-induced event preceding the onset of insulin resistance. Either genetic or pharmacological inhibition of v-ATPase in cardiomyocytes exposed to low palmitate concentrations reduced insulin sensitivity and cardiomyocyte contractility, which was rescued by CD36 silencing. The mechanism of palmitate-induced v-ATPase inhibition involved its dissociation into two parts: the cytosolic V-1 and the integral membrane V-0 subcomplex. Interestingly, oleate also inhibits v-ATPase function, yielding triacylglycerol accumulation but not insulin resistance. In conclusion, lipid oversupply increases CD36-mediated lipid uptake that directly impairs v-ATPase function. This feeds forward to enhanced CD36 translocation and further increased lipid uptake. In the case of palmitate, its accelerated uptake ultimately precipitates into cardiac insulin resistance and contractile dysfunction

Cardiac contractile dysfunction in insulin-resistant rats fed a high-fat diet is associated with elevated CD36-mediated fatty acid uptake and esterification

Changes in cardiac substrate utilisation leading to altered energy metabolism may underlie the development of diabetic cardiomyopathy. We studied cardiomyocyte substrate uptake and utilisation and the role of the fatty acid translocase CD36 in relation to in vivo cardiac function in rats fed a high-fat diet (HFD).Rats were exposed to an HFD or a low-fat diet (LFD). In vivo cardiac function was monitored by echocardiography. Substrate uptake and utilisation were determined in isolated cardiomyocytes.Feeding an HFD for 8 weeks induced left ventricular dilation in the systolic phase and decreased fractional shortening and the ejection fraction. Insulin-stimulated glucose uptake and proline-rich Akt substrate 40 phosphorylation were 41% (p <0.001) and 45% (p <0.05) lower, respectively, in cardiomyocytes from rats on the HFD. However, long-chain fatty acid (LCFA) uptake was 1.4-fold increased (p <0.001) and LCFA esterification into triacylglycerols and phospholipids was increased 1.4- and 1.5-fold, respectively (both p <0.05), in cardiomyocytes from HFD compared with LFD hearts. In the presence of the CD36 inhibitor sulfo-N-succinimidyloleate, LCFA uptake and esterification were similar in LFD and HFD cardiomyocytes. In HFD hearts CD36 was relocated to the sarcolemma, and basal phosphorylation of a mediator of CD36-trafficking, i.e. protein kinase B (PKB/Akt), was increased.Feeding rats an HFD induced cardiac contractile dysfunction, which was accompanied by the relocation of CD36 to the sarcolemma, and elevated basal levels of phosphorylated PKB/Akt. The permanent presence of CD36 at the sarcolemma resulted in enhanced rates of LCFA uptake and myocardial triacylglycerol accumulation, and may contribute to the development of insulin resistance and diabetic cardiomyopathy