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, FABPpm, 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.     

Contractions but not AICAR increase FABPpm content in rat muscle sarcolemma

In the present study, it was investigated whether acute muscle contractions in rat skeletal muscle increased the protein content of FABPpm in the plasma membrane. Furthermore, the effect of AICAR stimulation on FAT/CD36 and FABPpm protein content in sarcolemma of rat skeletal muscle was evaluated. Methods Male wistar rats (150 g) were anesthetized and either subjected to in situ electrically induced contractions (hindlimb muscles: 20 min, 10–20 V, 200 ms trains, 100 Hz) or stimulated with the pharmacological activator of AMPK, AICAR. To investigate changes in the content of FABPpm and FAT/CD36 in the plasma membrane by these stimuli, the giant sarcolemma vesicle (GSV) technique was applied. The hindlimb muscles were removed and used for the production of GSV and lysates. All samples were analyzed using the western blotting technique. Results Electrical stimulation of rat hindlimb muscle resulted in an increase in FABPpm protein content in the GSV of 61% (P < 0.05) and in FAT/CD36 protein content in the GSV of 33% (P < 0.05). AICAR stimulation increased FAT/CD36 protein content in GSV by 22% (P < 0.05), whereas FABPpm protein content in GSV was unaffected by AICAR treatment. There was no change in total FAT/CD36 and FABPpm protein expression, measured in lysates with western blotting, by either stimulus. AMPK thr172 and ERK1/2 thr202/204 phosphorylation were significantly increased with muscle contractions (P < 0.05), whereas only AMPK thr172 phosphorylation was increased with AICAR stimulation (P < 0.05). Conclusion These data show that contractions increase both FAT/CD36 and FABPpm protein content in skeletal muscle plasma membrane, whereas only FAT/CD36 protein content is increased when muscle are stimulated with AICAR. This suggests that AMPK is involved in regulation of FAT/CD36, but not FABPpm in skeletal muscle. However, since both ERK1/2 thr202/204 and AMPK thr172 phosphorylation are increased during muscle contractions, the present study cannot rule out that both could play a significant role in regulation of FAT/CD36 and FABPpm during muscle contractions.     

Modest PGC-1alpha overexpression in muscle in vivo is sufficient to increase insulin sensitivity and palmitate oxidation in subsarcolemmal, not intermyofibrillar, mitochondria

PGC-1alpha 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-1alpha overexpression in transgenic animals. Therefore, we examined the effects of a modest PGC-1alpha overexpression in a single rat muscle, in vivo, on fuel-handling proteins and insulin sensitivity. We also examined whether modest PGC-1alpha 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-1alpha 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, alpha2 subunit, and HSL. Transfection of PGC-1alpha into the red (RTA) and white tibialis anterior (WTA) compartments of the tibialis anterior muscle increased PGC-1alpha 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, alpha2 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-1alpha 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-1alpha overexpression up-regulated selected plasmalemmal and mitochondrial fuel-handling proteins, increased SS (not IMF) mitochondrial fatty acid oxidation, and improved insulin sensitivity.     

Regulation of fatty acid transport and membrane transporters in health and disease

Long chain fatty acid uptake across the plasma membrane occurs, in part, via a protein-mediated process involving a number of fatty acid binding proteins known as fatty acid transporters. A critical step in furthering the understandings of fatty acid transport was the discovery that giant vesicles, prepared from tissues such as muscle and heart, provided a suitable system for measuring fatty acid uptake. These vesicles are large (10–15 m diameter), are oriented fully right side out, and contain cytosolic FABP in the lumen, which acts as a fatty acid sink, while none of the fatty acid taken up is metabolized or associated with the plasma membrane. The key fatty acid transporters FAT/CD36 and FABPpm are expressed in muscle and heart and their plasma membrane content is positively correlated with rates of fatty acid transport. These transporters are regulated acutely (within minutes) and chronically (days). For instance, both muscle contraction and insulin can translocate FAT/CD36 from an intracellular pool to the plasma membrane, thereby increasing fatty acid transport. With obesity, fatty acid transport is increased along with a concomitant increase in plasmalemmal FAT/CD36 (heart, muscle) and FABPpm (heart only), but without change in the expression of these transporters. This latter observation suggests that some of the fatty acid transporters are permanently relocated to the plasma membrane. In other studies it also appears that fatty acid transport rates are altered in a reciprocal manner to glucose transport. Since disorders in lipid metabolism appear to be an important factor contributing to the etiology of a number of common human diseases such as diabetes and obesity, our evidence that protein-mediated fatty acid transport is a key step in lipid metabolism allows the speculation that malfunctioning of the fatty acid transport process could be a common critical factor in the pathogenesis of these diseases.     

Changes in fatty acid transport and transporters are related to the severity of insulin deficiency

We have examined the effects of streptozotocin (STZ)-induced diabetes (moderate and severe) on fatty acid transport and fatty acid transporter (FAT/CD36) and plasma membrane-bound fatty acid binding protein (FABPpm) expression, at the mRNA and protein level, as well as their plasmalemmal localization. These studies have shown that, with STZ-induced diabetes, 1) fatty acid transport across the plasma membrane is increased in heart, skeletal muscle, and adipose tissue and is reduced in liver; 2) changes in fatty acid transport are generally not associated with changes in fatty acid transporter mRNAs, except in the heart; 3) increases in fatty acid transport in heart and skeletal muscle occurred with concomitant increases in plasma membrane FAT/CD36, whereas in contrast, the increase and decrease in fatty acid transport in adipose tissue and liver, respectively, were accompanied by concomitant increments and reductions in plasma membrane FABPpm; and finally, 4) the increases in plasma membrane transporters (FAT/CD36 in heart and skeletal muscle; FABPpm in adipose tissue) were attributable to their increased expression, whereas in liver, the reduced plasma membrane FABPpm appeared to be due to its relocation within the cell in the face of slightly increased expression. Taken together, STZ-induced changes in fatty acid uptake demonstrate a complex and tissue-specific pattern, involving different fatty acid transporters in different tissues, in combination with different underlying mechanisms to alter their surface abundance.     

A null mutation in H-FABP only partially inhibits skeletal muscle fatty acid metabolism

The low-molecular-mass, cytosolic heart-type fatty acid-binding protein (H-FABP) is thought to be required for shuttling FA through the cytosol. Therefore, we examined the effects of an H-FABP-null mutation on FA and carbohydrate metabolism in isolated soleus muscle at rest and during a period of increased metabolic demand (30-min contraction). There were lower concentrations of creatine phosphate (-41%), ATP (-22%), glycogen (-34%), and lactate (-31%) (P < 0.05) in H-FABP-null soleus muscles, but no differences in citrate synthase and beta-3-hydroxyacyl-CoA dehydrogenase activities or in the intramuscular triacylglycerol (TAG) depots. There was a 43% increase in subsarcolemmal mitochondria in H-FABP-null solei. FA transport was reduced by 30% despite normal content of sarcolemmal long-chain fatty acid transporters fatty acid translocase/CD36 and plasma membrane-associated FABP transport proteins. Compared with wild-type soleus muscles, the H-FABP-null muscles at rest hydrolyzed less TAG (-22%), esterified less TAG (-49%), and oxidized less palmitate (-71%). The H-FABP-null soleus muscles retained a substantial capacity to increase FA metabolism during contraction (TAG esterification by +72%, CO2 production by +120%), although these rates remained lower (TAG esterification -26% and CO2 production -64%) than in contracting wild-type soleus muscles. Glycogen utilization during 30 min of contraction did not differ, whereas glucose oxidation was lower at rest (-24%) and during contraction (-32%) in H-FABP-null solei. Although these studies demonstrate that the absence of H-FABP alters rates of FA metabolism, it is also apparent that glucose oxidation is downregulated. The substantial increase in FA metabolism in contracting H-FABP-null muscle may indicate that other FABPs are also present, a possibility that we were not able to completely eliminate.     

Metabolic challenges reveal impaired fatty acid metabolism and translocation of FAT/CD36 but not FABPpm in obese Zucker rat muscle

We examined, in muscle of lean and obese Zucker rats, basal, insulin-induced, and contraction-induced fatty acid transporter translocation and fatty acid uptake, esterification, and oxidation. In lean rats, insulin and contraction induced the translocation of the fatty acid transporter FAT/CD36 (43 and 41%, respectively) and plasma membrane-associated fatty acid binding protein (FABPpm; 19 and 60%) and increased fatty acid uptake (63 and 40%, respectively). Insulin and contraction increased lean muscle palmitate esterification and oxidation 72 and 61%, respectively. In obese rat muscle, basal levels of sarcolemmal FAT/CD36 (+33%) and FABPpm (+14%) and fatty acid uptake (+30%) and esterification (+32%) were increased, whereas fatty acid oxidation was reduced (-28%). Insulin stimulation of obese rat muscle increased plasmalemmal FABPpm (+15%) but not plasmalemmal FAT/CD36, blunted fatty acid uptake and esterification, and failed to reduce fatty acid oxidation. In contracting obese rat muscle, the increases in fatty acid uptake and esterification and FABPpm translocation were normal, but FAT/CD36 translocation was impaired and fatty acid oxidation was blunted. There was no relationship between plasmalemmal fatty acid transporters and palmitate partitioning. In conclusion, fatty acid metabolism is impaired at several levels in muscles of obese Zucker rats; specifically, they are 1) insulin resistant with respect to FAT/CD36 translocation and fatty acid uptake, esterification, and oxidation and 2) contraction resistant with respect to fatty acid oxidation and FAT/CD36 translocation, but, conversely, 3) obese muscles are neither insulin nor contraction resistant at the level of FABPpm. Finally, 4) there is no evidence that plasmalemmal fatty acid transporters contribute to the channeling of fatty acids to specific metabolic destinations within the muscle.     

Polyethylene Glycol (PEG) Linked to Near Infrared (NIR) Dyes Conjugated to Chimeric Anti-Carcinoembryonic Antigen (CEA) Antibody Enhances Imaging of Liver Metastases in a Nude-Mouse Model of Human Colon Cancer

We report here that polyethylene glycol (PEG) linked to near infrared dyes conjugated to chimeric mouse-human anti-carcinoembryonic antigen (CEA) antibody greatly improves imaging of liver metastases in a nude mouse model of colon-cancer experimental metastases. PEGylated and non-PEGylated DyLight 650 and 750 dyes were conjugated to the chimeric anti-CEA antibody. The dyes were initially injected intravenously into nude mice without tumors. Tissue biodistribution was determined by tissue sonication and analyzing tissue dye concentration profiles over time. PEGylated dyes had significantly lower accumulation in the liver (p = 0.03 for the 650 dyes; p = 0.002 for the 750 dyes) compared to non-PEGylated dyes. In an experimental liver metastasis model of HT-29 colon cancer, PEGylated dyes conjugated to the anti-CEA antibody showed good labeling of metastatic tumors with high contrast between normal and malignant tissue which was not possible with the non-PEGylated dyes since there was so much non-specific accumulation in the liver. PEGylation of the DyLight 650 and 750 NIR dyes significantly altered tissue biodistribution, allowing brighter tissue labeling, decreased accumulation in normal organs, particularly the liver. This enabled high fidelity and high contrast imaging of liver metastases.     

Cellular lipid binding proteins as facilitators and regulators of lipid metabolism

Evidence is accumulating that cellular lipid binding proteins are playing central roles in cellular lipid uptake and metabolism. Membrane-associated fatty acid-binding proteins putatively function in protein-mediated transmembrane transport of fatty acids, likely coexisting with passive diffusional uptake. The intracellular trafficking of fatty acids, bile acids, and other lipid ligands, may involve their interaction with specific membrane or protein targets, which are unique properties of some but not of all cytoplasmic lipid binding proteins. Recent studies indicate that these proteins not only facilitate but also regulate cellular lipid utilization. For instance, muscle fatty acid uptake is subject to short-term regulation by translocation of fatty acid translocase (FAT)/CD36 from intracellular storage sites to the plasma membrane, and liver-type cytoplasmic fatty acid-binding protein (L-FABP_c) functions in long-term, ligand-induced regulation of gene expression by directly interacting with nuclear receptors. Therefore, the properties of the lipid-protein complex, rather than those of the lipid ligand itself, determine the fate of the ligand in the cell. Finally, there are an increasing number of reports that deficiencies or altered functioning of both membrane-associated and cytoplasmic lipid binding proteins are associated with disease states, such as obesity, diabetes and atherosclerosis. In conclusion, because of their central role in the regulation of lipid metabolism, cellular lipid binding proteins are promising targets for the treatment of diseases resulting from or characterised by disturbances in lipid metabolism, such as atherosclerosis, hyperlipidemia, and insulin resistance.