Development and initial evaluation of a novel method for assessing tissue-specific plasma free fatty acid utilization in vivo using (R)-2-bromopalmitate tracer.
We describe a method for assessing tissue-specific plasma free fatty acid (FFA) utilization in vivo using a non-beta-oxidizable FFA analog, [9,10-3H]-(R)-2-bromopalmitate (3H-R-BrP). Ideally 3H-R-BrP would be transported in plasma, taken up by tissues and activated by the enzyme acyl-CoA synthetase (ACS) like native FFA, but then 3H-labeled metabolites would be trapped. In vitro we found that 2-bromopalmitate and palmitate compete equivalently for the same ligand binding sites on albumin and intestinal fatty acid binding protein, and activation by ACS was stereoselective for the R-isomer. In vivo, oxidative and non-oxidative FFA metabolism was assessed in anesthetized Wistar rats by infusing, over 4 min, a mixture of 3H-R-BrP and [U-14C] palmitate (14C-palmitate). Indices of total FFA utilization (R*f) and incorporation into storage products (Rfs') were defined, based on tissue concentrations of 3H and 14C, respectively, 16 min after the start of tracer infusion. R*f, but not Rfs', was substantially increased in contracting (sciatic nerve stimulated) hindlimb muscles compared with contralateral non-contracting muscles. The contraction-induced increases in R*f were completely prevented by blockade of beta-oxidation with etomoxir. These results verify that 3H-R-BrP traces local total FFA utilization, including oxidative and non-oxidative metabolism. Separate estimates of the rates of loss of 3H activity indicated effective 3H metabolite retention in most tissues over a 16-min period, but appeared less effective in liver and heart. In conclusion, simultaneous use of 3H-R-BrP and [14C]palmitate tracers provides a new useful tool for in vivo studies of tissue-specific FFA transport, utilization and metabolic fate, especially in skeletal muscle and adipose tissue.
Oakes
N D
ND
Department of Pharmacology, Astra-Hässle AB, S-431 83 Mölndal, Sweden.
Kjellstedt
A
A
Forsberg
G B
GB
Clementz
T
T
Camejo
G
G
Furler
S M
SM
Kraegen
E W
EW
Olwegård-Halvarsson
M
M
Jenkins
A B
AB
Ljung
B
B
D000273:Adipose Tissue / Q000378:metabolism; D000818:Animals; D001667:Binding, Competitive; D001692:Biological Transport; D002250:Carbon Radioisotopes; D003066:Coenzyme A Ligases / Q000378:metabolism; D005230:Fatty Acids, Nonesterified / Q000097:blood* / Q000378:metabolism*; D007413:Intestinal Mucosa / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008297:Male; D018482:Muscle, Skeletal / Q000378:metabolism; D010168:Palmitates / Q000008:administration & dosage / Q000378:metabolism; D019308:Palmitic Acid / Q000097:blood / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D012709:Serum Albumin / Q000378:metabolism; D013237:Stereoisomerism; D014316:Tritium
D002250:Carbon Radioisotopes; D005230:Fatty Acids, Nonesterified; D010168:Palmitates; D012709:Serum Albumin; D014316:Tritium; C022776:2-bromopalmitate; D019308:Palmitic Acid; D003066:Coenzyme A Ligases