scholarly journals A sensitive immunoassay for rat fatty acid translocase (CD36) using phage antibodies selected on cell transfectants: abundant presence of fatty acid translocase/CD36 in cardiac and red skeletal muscle and up-regulation in diabetes

1999 ◽  
Vol 337 (3) ◽  
pp. 407 ◽  
Author(s):  
Maurice M.A.L. PELSERS ◽  
Jan T. LUTGERINK ◽  
Frans A. VAN NIEUWENHOVEN ◽  
Narendra N. TANDON ◽  
Ger J. VAN DER VUSSE ◽  
...  
1999 ◽  
Vol 337 (3) ◽  
pp. 407-414 ◽  
Author(s):  
Maurice M. A. L. PELSERS ◽  
Jan T. LUTGERINK ◽  
Frans A. van NIEUWENHOVEN ◽  
Narendra N. TANDON ◽  
Ger J. van der VUSSE ◽  
...  

The rat membrane protein fatty acid translocase (FAT), which shows sequence similarity to human CD36 (a membrane protein supposedly involved in a variety of membrane processes), is implicated in the transport of long-chain fatty acids across cellular membranes. To set up an immunoassay for quantification of FAT in different tissues, we isolated a series of anti-FAT antibodies by panning a large naive phage antibody library on FAT-transfected H9c2 cells. All seven different phage antibody fragments isolated reacted specifically with FAT, and most likely recognize the same or closely located immunodominant sites on FAT, as a competitive monoclonal antibody (mAb) (CLB-IV7) completely blocked the binding of all these phage antibodies to cells. A sandwich ELISA was set up using mAb 131.4 (directed against purified CD36 from human platelets) as capture antibody and phage antibodies and anti-phage sera as detector. With this ELISA (sensitivity 0.05 µg/ml), the FAT content in isolated cardiomyocytes was found to be comparable with that of total heart (≈ 3 mg/g of protein), while liver tissue and endothelial cells were below the detection limit (< 0.1 mg of FAT/g of protein). During rat heart development, protein levels of FAT rose from 1.7±0.7 mg/g of protein on the day before birth to 3.6±0.4 mg/g of protein on day 70. Comparing control with streptozotocin-induced diabetic rats, a statistically significant (P< 0.05) 2–4-fold increase of FAT was seen in heart (from 4.2±2.3 to 11.0±5.7 mg/g of protein), soleus (from 0.6±0.1 to 1.4±0.5 mg/g of protein) and extensor digitorum longus (EDL) muscle (from 0.3±0.1 to 1.2±0.8 mg/g of protein). In addition, the FAT contents of each of these muscles were found to be of similar magnitude to the contents of cytoplasmic heart-type fatty-acid-binding protein in both diabetic rats and controls, supporting the suggested roles of these two proteins in cellular fatty acid metabolism. This is the first time phage display technology has been succesfully applied for direct selection, from a large naive antibody library, of antibodies that recognize selected membrane proteins in their natural context.


2004 ◽  
Vol 65 (7) ◽  
pp. 951-956 ◽  
Author(s):  
Klien G. van Dam ◽  
Eric van Breda ◽  
Gert Schaart ◽  
Mireille M. E. van Ginneken ◽  
Inge D. Wijnberg ◽  
...  

2004 ◽  
Vol 75 (2) ◽  
pp. 155-159 ◽  
Author(s):  
Taiko BANDA ◽  
Dhanapati NEUPANEY ◽  
Jin-bo KIM ◽  
Makoto ISHIOROSHI ◽  
Katsuji MARUKAWA ◽  
...  

2004 ◽  
Vol 121 (2) ◽  
pp. 101-107 ◽  
Author(s):  
Hans A. Keizer ◽  
Gert Schaart ◽  
Narenda N. Tandon ◽  
Jan F. C. Glatz ◽  
Joost J. F. P. Luiken

2006 ◽  
Vol 290 (3) ◽  
pp. E509-E515 ◽  
Author(s):  
Veronic Bezaire ◽  
Clinton R. Bruce ◽  
George J. F. Heigenhauser ◽  
Narendra N. Tandon ◽  
Jan F. C. Glatz ◽  
...  

Fatty acid translocase (FAT/CD36) is a transport protein with a high affinity for long-chain fatty acids (LCFA). It was recently identified on rat skeletal muscle mitochondrial membranes and found to be required for palmitate uptake and oxidation. Our aim was to identify the presence and elucidate the role of FAT/CD36 on human skeletal muscle mitochondrial membranes. We demonstrate that FAT/CD36 is present in highly purified human skeletal mitochondria. Blocking of human muscle mitochondrial FAT/CD36 with the specific inhibitor sulfo- N-succimidyl-oleate (SSO) decreased palmitate oxidation in a dose-dependent manner. At maximal SSO concentrations (200 μM) palmitate oxidation was decreased by 95% ( P < 0.01), suggesting an important role for FAT/CD36 in LCFA transport across the mitochondrial membranes. SSO treatment of mitochondria did not affect mitochondrial octanoate oxidation and had no effect on maximal and submaximal carnitine palmitoyltransferase I (CPT I) activity. However, SSO treatment did inhibit palmitoylcarnitine oxidation by 92% ( P < 0.001), suggesting that FAT/CD36 may be playing a role downstream of CPT I activity, possibly in the transfer of palmitoylcarnitine from CPT I to carnitine-acylcarnitine translocase. These data provide new insight regarding human skeletal muscle mitochondrial fatty acid (FA) transport, and suggest that FAT/CD36 could be involved in the cellular and mitochondrial adaptations resulting in improved and/or impaired states of FA oxidation.


2008 ◽  
Vol 78 (2) ◽  
pp. 64-69 ◽  
Author(s):  
Choi ◽  
Cho

This study investigated the effect of vitamin B6 deficiency on the utilization and recuperation of stored fuel in physically trained rats. 48 rats were given either vitamin B6-deficient (B6–) diet or control (B6+) diet for 4 weeks and were trained on treadmill for 30 minutes daily. All animals were then subdivided into 3 groups: before-exercise (BE); during-exercise (DE); after-exercise (AE). The DE group was exercised on treadmill for 1 hour just before being sacrificed. Animals in the AE group were allowed to take a rest for 2 hours after being exercised like the DE group. Glucose and free fatty acids were compared in plasma. Glycogen and triglyceride were compared in liver and skeletal muscle. Protein levels were compared in plasma, liver, and skeletal muscle. Compared with the B6+ group, plasma glucose levels of the B6– group were significantly lower before and after exercise. Muscle glycogen levels of the B6– group were significantly lower than those of the B6+ group regardless of exercise. The liver glycogen level of the B6– group was also significantly lower than that of B6+ group during and after exercise. Before exercise, plasma free fatty acid levels were not significantly different between the B6+ and B6– groups, and plasma free fatty acid levels of the B6– group were significantly lower during and after exercise. The muscle triglyceride level of the B6– group was significantly lower than that of the B6+ group before exercise, and there were no differences between B6+ and B6– groups during and after exercise. Liver triglyceride levels were not significantly different between B6+ and B6– groups. Plasma protein levels of the B6– group were lower than those of B6+ before and after exercise. Muscle protein levels of the B6– group were not significantly different from those of the B6+ group. Liver protein levels of the B6– group were significantly lower than that of the B6+ group after exercise. Liver protein levels of both B6+ and B6– groups were not significantly changed, regardless of exercise. Thus, it is suggested that vitamin B6 deficiency may reduce fuel storage and utilization with exercise in physically trained rats.


2008 ◽  
Vol 194 (4) ◽  
pp. 293-309 ◽  
Author(s):  
G. P. Holloway ◽  
J. J. F. P. Luiken ◽  
J. F. C. Glatz ◽  
L. L. Spriet ◽  
A. Bonen

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