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Lipid synthesis and extraction. Cellular lipids were extracted essentially by the Folch method (6). Briefly, cells were incubated for 2 h at 37°C in the presence of [14C]acetic acid or [14C]glucose, washed once in cold PBS, and then lysed in 0.5 ml of ethanol. One milliliter of chloroform was added and mixed by vortexing. After addition of 0.5 ml of HCl (0.1 N), lysates were mixed by gentle inversion. Tubes were centrifuged at 500 g for 20 min. The upper phase and interface were discarded, and the organic phase was washed twice with 0.5 ml of water. Extracts were dried under N2, and the pellet was resuspended in the appropriate volume of chloroform-methanol (2:1). Extracts were separated by thin-layer chromatography with diethylether-hexane-glacial acetic acid (35:65:1, vol/vol/vol) as carrier solution on silica gel plates and exposed to film. Phospholipids and triacylglycerol were identified with standards.
from PMID 9081212
The cells were treated as described in the text and in the legend to Fig. 2, except the final concentration of acetate was 2 mM, at a specific activity of 0.1 mCi/mmol of [2-14C]-acetic acid, and was added to complete cell culture media containing 25 mM glucose. In each experiment, insulin resistant and control cell monolayers were assessed in parallel, and each experiment was performed at least four times. These assay condi- tions described for both glucose and acetate resulted in linear incorporation of radiolabel into cellular material for at least 60 rain. in both the presence and absence of insulin (data not shown).
Figure 2
The monolavers were incubated for 20 min in a CO2 incubator, followed by the addition of 2 ~uCi of [~4C]-glucose at a final specific activity of 0.05 mCi/mmol. The uptake and incorporation of radio- label was allowed to proceed for 20 rain, at 37 ° C, in the CO~ incubator, and was terminated by rapidly washing the monolayers with ice-cold phosphate- buffered saline (PBS). The monolayers were then scraped from each dish into 1.5 ml of PBS. and the cells were disrupted in a glass Dounce homogenizer. Neutral lipids were extracted from the cells into chlorofnrm:methanol:acetic acid, as previously described (Radin. 196o: Tarlow et ah, 1977). After evap- oration to dryness under nitrogen, the residue was dissolved in an organic solvent-based scintillation fluid and the radioactivity was quantitated by scin- tillation counting.
