Defining the role of DAG, mitochondrial function, and lipid deposition in palmitate-induced proinflammatory signaling and its counter-modulation by palmitoleate
Authors: Clare Stretton
Katherine Macrae
Marcin Baranowski
Christopher Lipina
Harinder S Hundal
Agnieszka Blachnio-Zabielska
Anna Marley
Jan Gorski
Journal: Journal of Lipid Research (2013)
Discipline: BIOLOGY & BIOCHEMISTRY
Keywords: cytokine; diacylglycerol; interleukin-6; monounsaturated fatty acid; nuclear factor kappa-B

Abstract:  Chronic exposure of skeletal muscle to saturated fatty acids, such as palmitate (C16:0), enhances proinflammatory IKK-NFκB signaling by a mechanism involving the MAP kinase (Raf-MEK-ERK) pathway. Raf activation can be induced by its dissociation from the Raf-kinase inhibitor protein (RKIP) by diacylglycerol (DAG)-sensitive protein kinase C (PKC). However, whether these molecules mediate the proinflammatory action of palmitate, an important precursor for DAG synthesis, is currently unknown. Here, involvement of DAG-sensitive PKCs, RKIP, and the structurally related monounsaturated fatty acid palmitoleate (C16:1) on proinflammatory signaling are investigated. Palmitate, but not palmitoleate, induced phosphorylation/activation of the MEK-ERK-IKK axis and proinflammatory cytokine (IL-6, CINC-1) expression. Palmitate increased intramyocellular DAG and invoked PKC-dependent RKIP(Ser153) phosphorylation, resulting in RKIP-Raf1 dissociation and MEK-ERK signaling. These responses were mimicked by PMA, a DAG mimetic and PKC activator. However, while pharmacological inhibition of PKC suppressed PMA-induced activation of MEK-ERK-IKK signaling, activation by palmitate was upheld, suggesting that DAG-sensitive PKC and RKIP were dispensable for palmitate's proinflammatory action. Strikingly, the proinflammatory effect of palmitate was potently repressed by palmitoleate. This repression was not due to reduced palmitate uptake but linked to increased neutral lipid storage and enhanced cellular oxidative capacity brought about by palmitoleate's ability to restrain palmitate-induced mitochondrial dysfunction. 

Retracted: No

PubPeer comments:
 Readers should become aware of a recent article entitled Beta-guanidinopropionic acid does not extend Drosophila lifespan published in Biochemistry and Biophysics Reports journal that was unable to reproduce the finding that Beta-guanidinopropionic acid can extend lifespan on flies. https: www.sciencedirect.com science article pii S2405580821001345?via%3Dihub#bib10 From the Discussion: Similar to Yang et al., our results demonstrate that GPA delivered in food in the range of 100 nMu2013100 mM is insufficient to extend lifespan or modify measures of functional aging in Drosophila. Moreover, we observed that 900 mM GPA, a dose previously suggested to increase lifespan, actually shortened median lifespan by 54% in males and 64% in females. In some ways, these results mirror recent reports published by the CITP [11] in which they found GPA had no effect on Caenorhabditis lifespan between 0.1 u03bcM and 10 mM, but 300 mM GPA significantly shortened the lifespan of C. briggsae. A similar pattern was observed in our experiments examining stress resistance with 100 mM GPA leading to reductions in both H2O2 and starvation resistance in male flies. Taken together, these results seem to suggest that dietary GPA supplementation in the 100 mM range and above may lead to significant toxicity in both flies and worms. (...) It is unclear why we were unable to replicate the findings regarding GPA and Drosophila lifespan previously reported by Yang et al. While the source of GPA used in these studies differs, our HPLC analyses shows our GPA source to be of high purity and similar to that sold by Sigma (median: 102.5%, intraquartile range (IQR): 97.2-106.7%; see methods). While we believe this supports the idea that both sources should be relatively equivalent in their effect, it is possible that other components, such as trace levels of containments, might drive the differences in our studies. However, as we discussed above the highest concentrations of GPA reportedly used in the Yang et al. study where much higher than we could solubilize in water or food during initial testing. While we were able to prepare food with 900 mM GPA, our results suggest this was extremely detrimental to male and female Drosophila. It is hard to reconcile these differences and it is unclear whether this is due to direct toxicity or indirect effects such as reduced palatability of the food and impairment of food intake. While we used the Yang et al. study as a guide for our own work, there are several potential differences between our studies that are not directly reported but might drive our disparate findings. For example, there are clear differences between our groups in the ability to solubilize GPA at high concentrations for food preparation. Differences in the homogeneity of GPA, or drug precipitation during food prep, might alter the distribution of this compound in the food which might then contribute to different outcomes between studies. Moreover, the process of food preparation and the potential degradation of GPA might drive these discrepancies in ways in which haven t yet been determined. Other potential external factors including food viscosity, relative humidity, final pH, microbial growth or microbiome makeup might also be plausible effectors of these differences that should likely be addressed. All these differences have been associated with changes in fly behavior and add to the complicated issue of replicating studies, particularly lifespan studies in different laboratories. While clarifying these differences is beyond the scope of this study, they all are potential important next steps to address in resolving the issue of what is the effect of GPA on lifespan. The work reported here highlights the challenges in replication of studies particularly of those addressing lifespan. However, by highlighting the differences in our studies we also highlight the lack of a robust effect of this intervention on Drosophila lifespan. Our results are however similar to the effect of GPA on lifespan reported in Caenorhabditis in that we found no effect on lifespan except reduction of lifespan at higher doses. It would be of interest to take a more standardized and thorough approach to test the effects of GPA, and other prolongevity compounds, across multiple species in an effort to identify robust interventions capable of modifying longevity. ,