Abstract
The FA composition of phospholipids must be tightly regulated to maintain optimal cell membrane properties and compensate for a highly variable supply of dietary FAs. Previous studies have shown that AdipoR2 and its homologue PAQR-2 are important regulators of phospholipid FA composition in HEK293 cells and Caenorhabditis elegans, respectively. Here we show that both AdipoR1 and AdipoR2 are essential for sustaining desaturase expression and high levels of unsaturated FAs in membrane phospholipids of many human cell types, including primary human umbilical vein endothelial cells, and for preventing membrane rigidification in cells challenged with exogenous palmitate, a saturated FA. Three independent methods confirm the role of the AdipoRs as regulators of membrane composition and fluidity: fluorescence recovery after photobleaching, measurements of Laurdan dye generalized polarization, and mass spectrometry to determine the FA composition of phospholipids. Furthermore, we show that the AdipoRs can prevent lipotoxicity in the complete absence of adiponectin, their putative ligand. We propose that the primary cellular function of AdipoR1 and AdipoR2 is to maintain membrane fluidity in most human cell types and that adiponectin is not required for this function.
Highlights
The FA composition of phospholipids must be tightly regulated to maintain optimal cell membrane properties and compensate for a highly variable supply of dietary FAs
We used siRNA to efficiently knock down the expression of AdipoR1 and AdipoR2 singly or simultaneously in HEK293 cells: Quantitative PCR (qPCR) confirmed the knockdown of the transcripts (Fig. 1A), and a Western blot confirmed protein downregulation for AdipoR1
Note that the Fluorescence recovery after photobleaching (FRAP) method measures the lateral diffusion rate of the BODIPY-C12 fluorophore used in these experiments; throughout this article we use the term “membrane fluidity” as a proxy for what is likely a complex phenomenon reflecting several distinct membrane biophysical properties such as fluidity, phase behavior, thickness, or compressibility [32,33,34]. siRNA knockdown of AdipoR1 had little effect on the membrane fluidity of HEK293 cells challenged with 200 M palmitic acid (PA), but inhibiting AdipoR2 caused a clear loss of membrane fluidity under these conditions (Fig. 1B, C), while inhibiting both AdipoR1 and AdipoR2 had the most dramatic effect (Fig. 1D, E)
Summary
The AdipoR1 and AdipoR2 proteins were initially identified as putative adiponectin receptors using fluorescentlabeled recombinant adiponectin as bait when screening a cDNA library expressed in Ba/F3 murine cells [1] These proteins are expressed in most/all tissues, localize to the plasma membrane, and contain seven transmembrane domains oriented such that their N terminus is cytosolic and their C terminus is extracellular [1]. Since their discovery, several high-profile reports using AdipoR1/2 single- or double-knockout mice have shown that the AdipoRs regulate metabolism and in particular may improve insulin response and generally protect against metabolic syndrome complications, especially during high-fat diet challenges [2,3,4].
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