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

• Published in: NMR in biomedicine Vol. 35; no. 3
• Main Authors: Patrício, João S., Dias‐Pedroso, Daniela, Carvalho, Rui A., Viera, Helena L. A., Jones, John G.
• Format: Journal Article
• Language: English
• Published: Oxford Wiley Subscription Services, Inc 01.03.2022
• Subjects: 13C‐isotopomer; Biological products; Cell culture; Enrichment; Fatty acids; Glucose; Glutamine; Lipid turnover; Lipids; Lipogenesis; Lipopolysaccharides; Microglia; NMR; Nuclear magnetic resonance; Precursors; Probabilistic models; Substrates
• ISSN: 0952-3480; 1099-1492
• DOI: 10.1002/nbm.4648

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 13C NMR analysis of lipid extracts from cultured microglia cells administered with [U‐13C]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‐13C]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 13C NMR. 13C‐isotopomer signals in the fatty acid ω − 1 and ω − 2 signals representing consecutive or non‐consecutive enrichment of the fatty acid chain by [1,2‐13C2]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 13C NMR analysis of the crude lipid fractions of BV‐2 cells administered with [U‐13C]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 13C NMR analysis approach to disclose the overall lipogenic activity and substrate contribution to DNL, suitable for evaluating DNL rates in cell cultures. In this article, we describe a novel and simple 13C NMR analysis approach to disclose the overall lipogenic activity and substrate contribution to DNL, suitable for evaluating DNL rates in cell cultures.