QUANTITATIVE ANALYSIS OF NADPH AND DE NOVO LIPOGENESIS

Abstract

Fat is one of the essential biomolecule categories. It is the major component of cell membrane and stores the most energy. Fat can either come directly from diet or be synthesized via de novo lipogenesis (DNL), a metabolic pathway converting carbohydrates into fat for long-term storage. Lipogenesis is important in both cell growth and energy metabolism, which makes it a druggable target in diseases like metabolic disorders and cancer. Lipogenesis needs acetyl-CoA as carbon building block and nicotinamide adenine dinucleotide phosphate (NADPH) as hydride donor. However, due to the instability and low abundance, the role of NADPH in lipogenesis was not fully understood, especially in vivo.To explore the source of NADPH, we developed LC-MS based analytical methods together with stable isotope tracing strategy to quantify NADPH production flux. We revealed that the active hydrogen on NADPH can exchange with water via a Flavin-enzyme catalyzed mechanism. Therefore, we built a parameter-free model to quantify both NADPH production and DNL flux. We confirmed that, in cultured mammalian cells, oxidative pentose phosphate pathway (oxPPP) produce the majority of cytosolic NADPH from glucose to make fat. We then applied this quantitative tracing strategy in vivo. We found that, in mammals, whole-body DNL is critical in producing saturated fatty acids. Liver and brown adipose tissue (BAT) have the highest turnover rate of fat. While BAT prefers directly catabolize glucose to generate both acetyl-CoA and NADPH, liver, however, prefer other sources. Unlike BAT, liver utilizes acetate and lactate as carbon source and serine as NADPH source. To further prove this point, we genetically and pharmaceutically inhibited serine catabolism in vivo, which resulted in the selective inhibition of hepatic lipogenesis. DNL is also up-regulated in uncontrolled replicating tumor cells. So, we examined whole-body DNL flux in tumor bearing mammals. Our results not only proved the increase DNL flux in Kras-driven tumors, but also showed a significant decrease in hepatic DNL, which may contribute to cancer cachexia. In summary, this study developed novel quantitative methods and discovered pathways supporting lipogenesis in liver, BAT, and cancer, which may shed light on the possibility of tissue-specific intervention of lipogenesis.