A simple method for quantifying de novo lipogenesis rate and substrate selection in cell cultures by ¹³C NMR isotopomer analysis of the crude lipid fraction

Purpose De novo lipogenesis (DNL) is critical for cell growth and maintenance, and acetyl-CoA precursors can be derived from different substrates. We developed a C-13 NMR analysis of lipid extracts from cultured microglia cells administered with [U-C-13]glucose that informs overall lipogenic activity as well as the contribution of glucose to lipogenic acetyl-CoA. Methods BV-2 microglial cell line cultured with glucose and glutamine was provided with [U-C-13]glucose and unlabeled glutamine for 24 h and studied in either the presence or absence of lipopolysaccharide (LPS). Cells were then extracted for lipids and the crude lipid fraction was analyzed by C-13 NMR. C-13-isotopomer signals in the fatty acid omega - 1 and omega - 2 signals representing consecutive or non-consecutive enrichment of the fatty acid chain by [1,2-C-13(2)]acetyl-CoA were quantified and applied to a probabilistic model of acetyl-CoA precursor and fatty acid enrichment. Results Glucose contributed 72 +/- 2% of lipogenic acetyl-CoA while DNL from all sources accounted for 16 +/- 2% of lipid turnover. With LPS, there was a significant decrease in glucose contribution (59 +/- 4%, p < 0.05) while DNL was unchanged (11 +/- 3%). Conclusions A simple C-13 NMR analysis of the crude lipid fractions of BV-2 cells administered with [U-C-13]glucose informs DNL activity and the contribution of glucose to the acetyl-CoA precursors. While DNL was preserved in the presence of LPS, there was redirection of lipogenic acetyl-CoA sources from glucose to other substrates. Thus, in the present article, we describe a novel and simple C-13 NMR analysis approach to disclose the overall lipogenic activity and substrate contribution to DNL, suitable for evaluating DNL rates in cell cultures.