Abstract
The branched chain amino acids (BCAA) valine, leucine and isoleucine have been implicated in a number of diseases including obesity, insulin resistance, and type 2 diabetes mellitus, although the mechanisms are still poorly understood. Adipose tissue plays an important role in BCAA homeostasis by actively metabolizing circulating BCAA. In this work, we have investigated the link between BCAA catabolism and fatty acid synthesis in 3T3-L1 adipocytes using parallel 13C-labeling experiments, mass spectrometry and model-based isotopomer data analysis. Specifically, we performed parallel labeling experiments with four fully 13C-labeled tracers, [U-13C]valine, [U-13C]leucine, [U-13C]isoleucine and [U-13C]glutamine. We measured mass isotopomer distributions of fatty acids and intracellular metabolites by GC-MS and analyzed the data using the isotopomer spectral analysis (ISA) framework. We demonstrate that 3T3-L1 adipocytes accumulate significant amounts of even chain length (C14:0, C16:0 and C18:0) and odd chain length (C15:0 and C17:0) fatty acids under standard cell culture conditions. Using a novel GC-MS method, we demonstrate that propionyl-CoA acts as the primer on fatty acid synthase for the production of odd chain fatty acids. BCAA contributed significantly to the production of all fatty acids. Leucine and isoleucine contributed at least 25% to lipogenic acetyl-CoA pool, and valine and isoleucine contributed 100% to lipogenic propionyl-CoA pool. Our results further suggest that low activity of methylmalonyl-CoA mutase and mass action kinetics of propionyl-CoA on fatty acid synthase result in high rates of odd chain fatty acid synthesis in 3T3-L1 cells. Overall, this work provides important new insights into the connection between BCAA catabolism and fatty acid synthesis in adipocytes and underscores the high capacity of adipocytes for metabolizing BCAA.
Highlights
MethodsMaterialsCulture materials were purchased from Cellgro (Mediatech, Manassas, VA). [U-13C]Valine (99 atom% 13C), [U-13C]leucine (98%), [U-13C,15N]isoleucine (99% 13C), [U-13C]glutamine (98%13C) and [U-13C]algal amino acids (97+% 13C) were purchased from Sigma-Aldrich
The branched chain amino acids (BCAA) valine, leucine and isoleucine are essential amino acids that comprise about 15–25% of total protein intake [1]
3T3-L1 cells were cultured from the pre-adipocyte state to confluence and 24 hours post confluence, the cells were chemically differentiated with insulin, dexamethasone and IBMX to achieve the mature adipocyte phenotype (Fig 1)
Summary
MaterialsCulture materials were purchased from Cellgro (Mediatech, Manassas, VA). [U-13C]Valine (99 atom% 13C), [U-13C]leucine (98%), [U-13C,15N]isoleucine (99% 13C), [U-13C]glutamine (98%13C) and [U-13C]algal amino acids (97+% 13C) were purchased from Sigma-Aldrich Culture materials were purchased from Cellgro (Mediatech, Manassas, VA). [U-13C]Valine (99 atom% 13C), [U-13C]leucine (98%), [U-13C,15N]isoleucine (99% 13C), [U-13C]glutamine 13C) and [U-13C]algal amino acids (97+% 13C) were purchased from Sigma-Aldrich (St. Louis, MO). Free amino acids were purchased from Fisher Sci. Amino acid standard H was purchased from Pierce Sci. Stock solutions (40x) of natural and uniformly 13C-labeled valine, leucine, and isoleucine were prepared at 40mM in deionized water. Stock solutions (40x) of natural and [U-13C]glutamine were prepared at 160mM in deionized water. [U-13C]algal amino acids were solubilized at 10mg/mL in 0.1N HCl phosphate buffered saline. All amino acid solutions were stored at -85°C until use
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