Transport of 13C-oleate in adipocytes measured using multi imaging mass spectrometry.
The mechanism of long chain free fatty acid (FFA) transport across cell membranes is under active investigation. Here we describe the use of multi imaging mass spectrometry (MIMS) to monitor intracellular concentrations of FFA and provide new insight into FFA transport in cultured adipocytes. Cells were incubated with 13C-oleate:BSA and either dried directly or dried after washing with a medium deprived of 13C-oleate:BSA. Cells were analyzed with MIMS using a scanning primary Cs+ ion beam and 12C-, 13C-, 12C14N-, 13C14N-) (or 12C 15N-) were imaged simultaneously. From these quantitative images the values of the 13C/ 12C ratios were determined in the intracellular lipid droplets, in the cytoplasm and outside the 3T3F442A adipocytes. The results indicate that after incubation with 13C-oleate:BSA the droplet 13C/ 12C ratio was 15 +/- 6%. This value is about 14-fold higher than the 13C/ 12C terrestrial ratio (1.12%). After washing the 13C-oleate:BSA, the droplet 13C/ 12C ratios decreased to 1.6 +/- 0.1%, about 40% greater than the natural abundance. Results for washed cells indicate that relatively little FFA was esterified. The unwashed cell results, together with the value of the lipid water partition coefficient, reveal that intracellular unbound FFA (FFAu) concentrations were on average about 4.5-fold greater than the extracellular FFAu concentrations. These results are consistent with the possibility that FFA may be pumped into adipocytes against their electro-chemical potential. This work demonstrates that MIMS can be used to image and quantitate stable isotope labeled fatty acid in intracellular lipid droplets.
Kleinfeld
Alan M
AM
Torrey Pines Institute for Molecular Studies, San Diego, California 92121, USA. akleinfeld@tpims.org
Kampf
J Patrick
JP
Lechene
Claude
C
D016475:3T3 Cells; D017667:Adipocytes / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D001692:Biological Transport; D002247:Carbon Isotopes; D005230:Fatty Acids, Nonesterified / Q000032:analysis / Q000378:metabolism; D007091:Image Processing, Computer-Assisted; D051379:Mice; D019301:Oleic Acid / Q000493:pharmacokinetics*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000295:instrumentation* / Q000379:methods*
D002247:Carbon Isotopes; D005230:Fatty Acids, Nonesterified; D019301:Oleic Acid