Subcellular Localization and Role of Lipin-1 in Human Macrophages

Abstract

The lipins have been described as metabolic enzymes that regulate lipid biosynthesis and also signaling processes by controlling the cellular concentration of bioactive lipids, phosphatidic acid, and diacylgycerol. In the present work we have studied the subcellular localization and role of lipin-1 in human monocyte-derived macrophages. Human macrophages express lipin-1 isoforms α and β. A transfected lipin-1α–enhanced GFP construct associates with membranes of cellular organelles that can be stained with Nile Red. Colocalization experiments with lipid droplet (LD)-specific proteins such as adipophilin/adipose differentiation-related protein/perilipin 2 or TIP47/perilipin 3 show that both proteins colocalize with lipin-1α in the same cellular structures. Reduction of the expression levels of lipin-1 by small interfering RNA technology does not impair triacylglycerol biosynthesis but reduces the size of LDs formed in response to oleic acid. In agreement with these data, peritoneal macrophages from animals that carry a mutation in the Lpin-1 gene (fld animals) also produce less and smaller LDs in response to oleic acid. Mass spectrometry determinations demonstrate that the fatty acid composition of triacylglycerol in isolated LDs from lipin-1–deficient cells differs from that of control cells. Moreover, activation of cytosolic group IVA phospholipase A2α, a proinflammatory enzyme that is also involved in LD biogenesis, is also compromised in lipin-1–deficient cells. Collectively, these data suggest that lipin-1 associates with LDs and regulates the activation of cytosolic group IVA phospholipase A2α in human monocyte-derived macrophages.

Lipin-2 Reduces Proinflammatory Signaling Induced by Saturated Fatty Acids in Macrophages

“…However, TAG synthesis itself does not appear to be regulated by lipin-1, because depletion of this protein does not affect the rate of fatty acid incorporation into TAG (20). In this study, we have observed that TAG mass in lipin-2-depleted macrophages, either human or murine, is significantly decreased as compared with cells expressing normal lipin-2 levels, and this occurs under normal or fatty acid overload conditions.…”

“…In previous work, we demonstrated that lipin-1 modulates size, amount, and TAG fatty acid composition of lipid droplets in human macrophages (20). However, TAG synthesis itself does not appear to be regulated by lipin-1, because depletion of this protein does not affect the rate of fatty acid incorporation into TAG (20).…”

“…Flow Cytometry-After the various treatments, cells were scraped, washed with phosphate-buffered saline, and stained with 0.2 g/ml BODIPY 493/503 for 5 min as described previously (20). Cells were washed, and fluorescence was analyzed by flow cytometry in a FACS Gallios (Beckman Coulter) using the FL1 detector.…”

“…Also, the siRNAs produced a 70 -90% reduction in the expression of the corresponding targets. Transfection of human macrophages was achieved by the Nucleofection method as described previously (20).…”

Identification of a Novel Cell-Penetrating Peptide from Human Phosphatidate Phosphatase LpIN3

“…Most previous studies about LPINs involved lipid biosynthesis and its related roles in adipose tissue or, more recently, in macrophages (Valdearcos et al, 2011). Here, we have suggested a previously unknown function of amino acids derived from LPIN3 protein by identifying and characterizing a cell-penetrating peptide, which is located on its nuclear localization sequence.…”