Abstract
Oxidative imbalance plays an essential role in the progression of many diseases that include cancer and neurodegenerative diseases. Aromatic amino acids (AAA) such as phenylalanine and tryptophan have the capability of escalating oxidative stress because of their involvement in the production of Reactive Oxygen Species (ROS). Here, we use D2O (heavy water) probed stimulated Raman scattering microscopy (DO-SRS) and two Photon Excitation Fluorescence (2PEF) microscopy as a multimodal imaging approach to visualize metabolic changes in HeLa cells under excess AAA such as phenylalanine or trytophan in culture media. The cellular spatial distribution of de novo lipogenesis, new protein synthesis, NADH, Flavin, unsaturated lipids, and saturated lipids were all imaged and quantified in this experiment. Our studies reveal ∼10% increase in de novo lipogenesis and the ratio of NADH to flavin, and ∼50% increase of the ratio of unsaturated lipids to saturated lipid in cells treated with excess phenylalanine or trytophan. In contrast, these cells exhibited a decrease in the protein synthesis rate by ∼10% under these AAA treatments. The cellular metabolic activities of these biomolecules are indicators of elevated oxidative stress and mitochondrial dysfunction. Furthermore, 3D reconstruction images of lipid droplets were acquired and quantified to observe their spatial distribution around cells’ nuceli under different AAA culture media. We observed a higher number of lipid droplets in excess AAA conditions. Our study showcases that DO-SRS imaging can be used to quantitatively study how excess AAA regulates metabolic activities of cells with subcellular resolution in situ.
Highlights
The aromatic amino acids (AAA), L-phenylalanine and L-tryptophan, are essential for protein synthesis (Parthasarathy et al, 2018), and serve as functional components in the regulation of many metabolic pathways (Wu, 2013) with implications in diseases such as cancer (Kimura and Watanable, 2016; Cheng et al, 2019; Ma et al, 2021)
With lipid droplets (LDs) being observed, we noticed that both 15x phenylalanine and 15x tryptophan show higher intensities of biomolecules compared to the control group at the 2143 cm−1 peak
This verifies that phenylalanine and tryptophan do have a notable effect on lipid metabolism in cancer cells, Principal component analysis (PCA) and t-SNE plots have limited ability to isolate specific peaks that contribute to major variances on Raman spectra in this study
Summary
The aromatic amino acids (AAA), L-phenylalanine and L-tryptophan, are essential for protein synthesis (Parthasarathy et al, 2018), and serve as functional components in the regulation of many metabolic pathways (Wu, 2013) with implications in diseases such as cancer (Kimura and Watanable, 2016; Cheng et al, 2019; Ma et al, 2021). The regulation of AAAs has the potential to amplify oxidative stress during the onset and progression of diseases This is because excess AAA, such as phenylalanine and tryptophan, can induce the production of Reactive Oxygen Species (ROS) by activating the mammalian target of rapamycin (mTOR) and promoting oxygen consumption and mitochondrial metabolism (Wang et al, 2015; Saxton and Sabatini, 2017a; Mossmann et al, 2018). With the failure of autophagy and lipophagy initiation, an accumulation of lipid droplets (LDs) and increase in ROS within cells can perhaps be observed and quantified These altered metabolic activities can contribute to mitochondrial dysfunctions that lead to the production of malignant precursors from healthy cells (Porporato et al, 2018). Due to the lack of non-invasive, label-free imaging methods, the role of AAA, specfically phenylalanine and trytophan, in cellular metabolism such as lipid synthesis and protein synthesis is unclear
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