Effect of Palmitate on Mitochondrial Function and the Implications on Inflammatory Pathway NLRP3/IL-1β in Mouse and Human Myeloid Cell Lines

Abstract

Type 2 Diabetes Mellitus (T2DM) and obesity has been increasing globally, and as a consequence they cause major public health problems. Chronic metabolic inflammation, common in obese and T2DM subjects, can be caused by excess of nutrients. The “NLRP3 inflammasome” the inflammatory pathway responsible for the secretion of the cytokines interleukin- (IL-) 1β and IL-18, has been linked to chronic metabolic inflammation. Overstimulation of the NLRP3 inflammatory pathway is connected to several chronic diseases related to obesity, such as type II diabetes, and cardiovascular disease. There is a consensus that mitochondrial reactive oxygen species (ROS) are elevated during NLRP3 inflammasome activation. The goal of this study was to investigate the oxidative stress and inflammation associated with fatty acid overload in mouse and human myeloid cells. To mimic nutritional excess and induce inflammation, J774A.1 (mouse macrophage-like) or THP-1 (human monocytes) cells were treated with 2µM to 100µM of palmitate in presence or absence of LPS stimulation. Intracellular levels of the inflammasome proteins (NLRP3 and Pro-IL-1β) and the antioxidant enzymes (PRXSO3, SOD, Trx2) were analyzed by western blotting. Mitochondria derived ROS and overall intracellular ROS were analyzed using the fluorescent probes MitoSox and H2DCFDA, respectively, followed by cell imaging. In J774A.1 cells palmitate (2 to 10 μM) alone caused an increase in the level of the inflammatory proteins, NLRP3 and pro-IL-1β as well as mitochondrial ROS, but not overall intracellular ROS. Increases on IL-1 β secretion is only seen at higher concentration in J774A.1, while in THP-1 this effect appeared in lower doses as well. Overall, our results indicate that lower doses of palmitate induce increases on mitochondrial ROS production which may be associated with increases in downstream inflammatory response.