Abstract
Controversial correlations between biological activity and concentration of the novel lipokine palmitoleate (9Z-hexadecenoate, 16:1) might depend on the formation of an active 16:1 metabolite. For its identification, we analyzed the glycerophospholipid composition of mouse Swiss 3T3 fibroblasts in response to 16:1 using LC-MS/MS. 16:1 was either supplemented to the cell culture medium or endogenously formed when cells were stimulated with insulin or growth factors as suggested by the enhanced mRNA expression of 16:1-biosynthetic enzymes. The proportion of 1-acyl-2-16:1-sn-phosphatidylinositol (16:1-PI) was time-dependently and specifically increased relative to other glycerophospholipids under both conditions and correlated with the proliferation of fatty acid (16:1, palmitate, oleate, or arachidonate)-supplemented cells. Accordingly, cell proliferation was impaired by blocking 16:1 biosynthesis using the selective stearoyl-CoA desaturase-1 inhibitor CAY10566 and restored by supplementation of 16:1. The accumulation of 16:1-PI occurred throughout cellular compartments and within diverse mouse cell lines (Swiss 3T3, NIH-3T3, and 3T3-L1 cells). To elucidate further whether 16:1-PI is formed through the de novo or remodeling pathway of PI biosynthesis, phosphatidate levels and lyso-PI-acyltransferase activities were analyzed as respective markers. The proportion of 16:1-phosphatidate was significantly increased by insulin and growth factors, whereas lyso-PI-acyltransferases showed negligible activity for 16:1-coenzyme A. The relevance of the de novo pathway for 16:1-PI biosynthesis is supported further by the comparable incorporation rate of deuterium-labeled 16:1 and tritium-labeled inositol into PI for growth factor-stimulated cells. In conclusion, we identified 16:1 or 16:1-PI as mitogen whose biosynthesis is induced by growth factors.
Highlights
Conflicting reports about the function of palmitoleate might depend on the formation of bioactive metabolites
Using comprehensive liquid chromatography tandem mass spectrometry (LC-MS/MS)-based lipidomics, we identified 1-acyl-2–16:1-sn-phosphatidylinositol (16:1-PI) as strongly and regulated membrane PL. 16:1-PI is formed in response to exogenous 16:1 as well as to metabolic hormones and growth factors through induction of fatty acid and de novo PI biosynthesis
We found that 16:1 is synthesized upon stimulation with growth factors, induces cell proliferation, and is selectively incorporated into PI through de novo PI biosynthesis
Summary
Conflicting reports about the function of palmitoleate might depend on the formation of bioactive metabolites. The increase of 16:1 was found to protect against insulin resistance in liver and skeletal muscle by regulating a subset of metabolic enzymes [11] Along these lines, cellular studies found that 16:1 increases basal glucose uptake and activates insulin signaling [11,12,13], promotes -cell proliferation and secretory function (14 –16), regulates the expression and degradation of metabolic enzymes [11], and prevents 16:0-induced endoplasmatic reticulum stress and apoptosis [17, 18]. Cellular studies found that 16:1 increases basal glucose uptake and activates insulin signaling [11,12,13], promotes -cell proliferation and secretory function (14 –16), regulates the expression and degradation of metabolic enzymes [11], and prevents 16:0-induced endoplasmatic reticulum stress and apoptosis [17, 18] These unique characteristics of 16:1 among fatty acids led to the concept of 16:1 as lipid hormone coordinating metabolic responses [11]. Using comprehensive liquid chromatography tandem mass spectrometry (LC-MS/MS)-based lipidomics, we identified 1-acyl-2–16:1-sn-phosphatidylinositol (16:1-PI) as strongly and regulated membrane PL. 16:1-PI is formed in response to exogenous 16:1 as well as to metabolic hormones and growth factors through induction of fatty acid and de novo PI biosynthesis
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