Amyotrophic lateral sclerosis (ALS) is a type of motor neuron disease that results in paralysis and death from a progressive loss of both upper and lower motor neurons. Emerging studies have indicated that excess production of reactive oxygen species (ROS) in dysfunctional mitochondria in addition to an inefficient antioxidant defense may contribute to the progression of the disease. L-methionine (Met) plays important roles in regulating cellular metabolism and activating endogenous antioxidant enzymes. We hypothesized that excess methionine treatment can provide ALS patients with an efficient antioxidant defense mechanism to control their disease. Here, we apply an optical imaging approach that combines deuterium oxide (D2O)-probed stimulated Raman scattering (DO-SRS) and two photon excitation fluorescence (2PEF) microscopies to directly image the effects of methionine-enriched diet on oxidative imbalance and cellular metabolism in neurodegenerative cells. Our preliminary data revealed that excess methionine increases syntheses of lipid and unsaturated lipid membranes. Meanwhile, the same diet decreases protein synthesis and oxidative imbalance. Our study suggests that excess methionine can provide a protective mechanism against oxidative imbalance and promote cellular repair in neurodegenerative diseases.
We use D2O probed stimulated Raman scattering (DO-SRS) and Multiphoton Fluorescence (MPF) microscopy to visualize metabolic changes in HeLa cells under excess AAA of phenylalanine or tryptophan. The cellular spatial distribution of de novo lipogenesis, protein synthesis, NADH, Flavin, unsaturated lipids, and cholesterol were all imaged and quantified in this experiment. Our studies reveal the increase in NADH to Flavin ratio by 10% and unsaturated lipids to saturated by 50% in cells treated with excess phenylalanine and tryptophan. Our study shows that DO-SRS can be used to as a high resolution imaging platform to study AAA regulated metabolic activities in cells.
Emerging studies have shown that oxidative imbalance is critical in disease progression such as cancer and Alzheimer’s [1, 2]. This variation can lead to the upregulation of certain metabolic pathways inducing diseases and disorders. Aromatic amino acids (AAA) are involved with the production of Reactive Oxygen Species (ROS), resulting in the increase of oxidative stress [3]. AAA studies typically rely on gas chromatography (GC) or mass spectroscopy (MS)-based imaging techniques to study lipids; however, these methods lack the ability to show the cell’s lipid spatial distribution or require fluorescent dyes that can interfere with the cell’s molecular activities [4, 5]. Here, we established an optical imaging approach that combines D2O (heavy water) probed Stimulated Raman scattering (DO-SRS) and Multiphoton Fluorescence (MPF) microscopy to directly visualize metabolic activities in situ in cancer cells under the regulation of excess AAA, specifically Phenylalanine and Tryptophan. The cellular spatial distribution of de novo lipogenesis, unsaturated and saturated lipids, NADH, Flavin, and new protein synthesis were quantitatively imaged and examined. We discovered an increase in de novo lipogenesis, Flavin/(Flavin + NADH), and unsaturated to saturated lipids in the cancer cells treated with excess AAAs. Decrease of protein turnover rate occurred in the same treated cells with observations of higher lipid droplet content. These observed metabolic activities are signs of mitochondrial dysfunction and oxidative stress. Our study demonstrates that DO-SRS can be used as a high-resolution imaging platform to study AAA regulated metabolic activities in cells and elucidates the linkage between lipid metabolism and cancer.
Two breast cancer subtypes with contrasting aggressiveness and prognoses - triple negative and normal-like - had their lipid droplets interrogated under methionine and insulin manipulation. Raman scattering results suggest that excess methionine increases droplet volume and de novo lipogenesis but decreases droplet number and alters distribution in both subtypes. These effects are significantly augmented in the triple-negative subtype with the presence of insulin. Flavin autofluorescence increases with insulin but decreases with excess methionine, in which an abundance of unsaturated lipids was also observed. This suggests interplay between methionine and insulin related lipid homeostasis and demonstrates the utility of optical techniques.
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