Branched fatty acid esters of hydroxy fatty acids (FAHFAs) are structurally novel, bioactive lipids that are synthesized in humans, animals, and plants and that have beneficial metabolic and anti-inflammatory effects (Kuda et al., 2016; Yore et al., 2014; Zhu et al., 2018). An in silico analysis predicted that> 1,000 FAHFAs could exist in nature (Ma et al., 2015), and> 20 FAHFA families have been identified in mammalian tissues (Kuda et al., 2016, 2018; Ma et al., 2015; Yore et al., 2014), with many additional families in plants (Zhu et al., 2018). These families differ by their acyl-chain composition and consist of multiple isomers distinguished by the position of the ester bond between acyl chains. Palmitic acid esters of hydroxy stearic acids (PAHSAs) have been studied most extensively. Levels of multiple PAHSA isomers are reduced in serum and subcutaneous adipose tissue of insulin-resistant mice and humans, and serum PAHSA levels correlate highly with insulin sensitivity in humans (Yore et al., 2014). A single oral dose of 5-PAHSA or 9-PAHSA improves glucose tolerance in aged, glucose-intolerant chow-fed and high-fat diet (HFD)-fed mice (Yore et al., 2014), whereas chronic subcutaneous PAHSA treatment improves both insulin sensitivity and glucose tolerance in chow-fed and HFD-fed mice (Syed et al., 2018). The anti-inflammatory effects of PAHSAs are seen in mouse models of insulin resistance (Syed et al., 2018; Vijayakumar et al., 2017; Yore et al., 2014), colitis (Lee et al., 2016), and type 1 diabetes (Syed et al., 2017).

Pflimlin et al.(2018) recently challenged the finding that PAHSAs have beneficial metabolic effects. We appreciate the attempt to corroborate our findings as reproducibility of results by different laboratories is critical. While these different results may be perceived as a reproducibility issue, numerous methodological differences, some of which were mentioned by Pflimlin et al.(2018) in their ‘‘limitations’’section, can explain why Pflimlin et al.’s experiments did not detect anti-diabetic effects (Tables S1 and S2). None of the experiments in Pflimlin et al. repeated the protocols used in any previous PAHSA/FAHFA papers.