Metabolism and structure of triacylglycerols in rat epididymal fat pad adipocytes determined by 13C nuclear magnetic resonance

Abstract

Carbon-13 nuclear magnetic resonance (NMR) methods have been applied to a study of the structure and metabolism of the triacylglycerols from rat epididymal fat pad adipocytes. Complete NMR signal assignments are provided for adipocytes, the extracted triacylglycerols, and methyl esters of the derived fatty acids. 13C NMR yielded rapid, nondestructive, quantitative analysis of the amounts of unsaturation of the fatty acyl chains; in cells from rats given ad libitum access to a standard laboratory diet the predominant fatty acids were found to be palmitate (29.9%), oleate (27.9%), and linoleate (34.1%). These results agreed with gas chromatographic separation of the derived methyl esters of the extracted lipids. Lipid dynamics were examined in situ and showed a substantial restriction of motion of glyceride-glycerol as compared with free glycerol; the nuclear magnetic spin-lattice relaxation times for free glycerol of 2.52 +/- 0.12 (C1,3) and 4.37 +/- 0.21 (C2) s decreased to 0.15 +/- 0.009 and 0.21 +/- 0.013 s, respectively, upon esterification. Segmental motion of the chains, monitored by relaxation time measurements, increased progressively from the alpha-carbon (nT1 = 0.70 s) to the methyl ends of the chains (nT1 = 9.63). The incorporation of C-13-labeled substrates ([1-13C]glucose and [3-13C]lactate) into the glycerol moiety of triacylglycerols was monitored in real time, in the presence of insulin. Lactate (10 mM) inhibited the incorporation of glucose (5.5 mM) into glyceride-glycerol. Lipolysis at the natural abundance level of 13C was measured in the presence of 10 microM isoproterenol. Simultaneous lipogenesis and lipolysis were found to occur in situ and were measured with the aid of [1-13C]glucose and isoproterenol; the labeling pattern of medium glycerol versus extracted triacylglycerols was significantly different from that found using natural abundance glucose. Our results indicate that 13C NMR is a useful new method for the real-time monitoring of lipid structure and metabolism in vivo.