(158g) Integrative Analysis of Free Fatty Acid-Induced NF?B Activation in Macrophages: A Step Towards the Quantitative Understanding of Inflammation Induced By Obesity

Over the past several decades, the obesity rate among U.S. adults has increased from 18% in 1998 to nearly 40% in 2015, which makes it one of the most prevalent public health issues in U.S. Caused by excess nutrition intake coupled with a lack of physical exercise, obesity greatly increases the risks of various diseases such as type 2 diabetes and cardiovascular diseases [1-3]. One of the proposed mechanisms linking obesity to these diseases is saturated fatty acids (SFA)-induced inflammation, which is characterized by the sustained production of pro-inflammatory cytokines such as tumor necrosis factor-α (TNFα) [3, 4]. Specifically, the elevated level of free SFA due to the obesity enhances the production of TNFα by macrophages in adipose tissues [3].

Previous studies have suggested two mechanisms that are important in the SFA-induced TNFα production. First, SFA activates Toll-like receptors (TLRs) that initiate the canonical nuclear factor-κB (NFκB) signaling pathway to synthesize TNFα [5]. Second, SFA dephosphorylates 5' adenosine monophosphate-activated protein kinase (AMPK) through a series of posttranslational modification (PTM) processes. And, the AMPK dephosphorylation enhances the TNFα synthesis [4]. However, the exact mechanisms still remain elusive since the roles of components involved in the mechanisms as well as their interactions are not clear yet. For example, recent studies [6, 7] raised questions about the roles of TLRs since they found TLRs were not involved in the SFA-induced NFκB activation in macrophages.

In this study, we used an integrative approach, which combines both experimental and computational methods, to investigate the TNFα production in macrophages stimulated by palmitate, which is the most common SFA in the body [8]. First, a dynamic model was constructed by incorporating the mechanisms suggested by the literature to predict the kinetics of the TNFα production in macrophages. Second, the dynamics of the TNFα expression and AMPK phosphorylation in RAW264.7 macrophages were measured by the time-resolved qRT-PCR immunoblotting upon the palmitate stimulation. And, the measurements were subsequently used to train and validate the proposed model. Third, in order to assess how each subprocess temporally influences the TNFα production, in silico knockout and perturbation studies were performed using the trained model. Specifically, a set of model parameters relevant to a subprocess of interest would be removed so that its effects on the TNFα production dynamics could be quantified. Subsequently, a new set of experiments were designed to verify the in silico studies. Through the integrative approach, the roles of TLRs as well as PTM enzymes such as protein phosphatase 2A, which regulate the AMPK activity, would be evaluated and discussed. In summary, this study gave quantitative insights into the molecular mechanisms of how palmitate induces the TNFα synthesis through the systematic framework.

References:

[1] Centers for Disease Control and Prevention. Adult Obesity Facts [Internet]. http://www.cdc.gov/obesity/data/adult.html (accessed April 4, 2019)

[2] Manteiga, S.; Choi, K.; Jayaraman, A.; Lee, K. Systems biology of adipose tissue metabolism: regulation of growth, signaling and inflammation, WIREs Systems Biology and Medicine 2013, 5, 425-447.

[3] Tateya, S.; Kim, F.; Tamori, Y. Recent advances in obesity-induced inflammation and insulin resistance, Frontiers in Endocrinology 2013, 4, 93.

[4] Yang, Z.; Kahn, B. B.; Shi, H.; Xue, B. Macrophage α1 AMP-activated Protein Kinase (α1AMPK) Antagonizes Fatty Acid-induced Inflammation through SIRT1, Journal of Biological Chemistry 2010, 285, 19051-19059.

[5] Huang, S.; Rutkowsky, J.M.; Snodgrass, R.G.; Ono-Moore, K.D.; Schneider, D.A.; Newman, J.W.; Adams, S. H.; Hwang, D. H. Saturated fatty acids activate TLR-mediated proinflammatory signaling pathways, Journal of Lipid Research 2012, 53, 2002–2013.

[6] Lancaster, G.I. et al. Evidence that TLR4 is not a receptor for saturated fatty acids but mediates lipid-induced inflammation by reprogramming macrophage metabolism. Cell Metabolism 2018, 27, 1096-1110.

[7] One-Moore, K. D.; Blackburn, M. L.; Adams, S. H. Is palmitate truly proinflammatory? Experimental confounders and context-specificity. American Journal of Physiology-Endocrinology and Metabolism 2018, 315, E780-E794.

[8] Carta, G.; Murru, E.; Banni, S.; Manca, C. Palmitic Acid: Physiological Role, Metabolism and Nutritional Implications, Frontiers in Physiology 2017, 8, 902.