Adaptations of the 3T3-L1 adipocyte lipidome to defective ether lipid catabolism upon Agmo knockdown

Abstract

Little is known about the physiological role of alkylglycerol monooxygenase (AGMO), the only enzyme capable of cleaving the 1-O-alkyl ether bond of ether lipids. Expression and enzymatic activity of this enzyme can be detected in a variety of tissues including adipose tissue. This labile lipolytic membrane-bound protein uses tetrahydrobiopterin as a cofactor, and mice with reduced tetrahydrobiopterin levels have alterations in body fat distribution and blood lipid concentrations. In addition, manipulation of AGMO in macrophages led to significant changes in the cellular lipidome, and alkylglycerolipids, the preferred substrates of AGMO, were shown to accumulate in mature adipocytes. Here, we investigated the roles of AGMO in lipid metabolism by studying 3T3-L1 adipogenesis. AGMO activity was induced over 11 days using an adipocyte differentiation protocol. We show that RNA interference-mediated knockdown of AGMO did not interfere with adipocyte differentiation or affect lipid droplet formation. Furthermore, lipidomics revealed that plasmalogen phospholipids were preferentially accumulated upon Agmo knockdown, and a significant shift toward longer and more polyunsaturated acyl side chains of diacylglycerols and triacylglycerols could be detected by mass spectrometry. Our results indicate that alkylglycerol catabolism has an influence not only on ether-linked species but also on the degree of unsaturation in the massive amounts of triacylglycerols formed during in vitro 3T3-L1 adipocyte differentiation.

Keywords: 3T3-L1; adipocyte differentiation; adipocytes; alkylglycerol monooxygenase; ether lipids; lipid metabolism; lipidomics; lipids; lipolysis and fatty acid metabolism; triacylglycerol.

Fig. 1

Determination of adipocyte-specific markers and AGMO activity during 3T3-L1 adipocyte differentiation. A: Oil Red O staining of differentiated (day 11, upper panel) and undifferentiated (day 0, lower panel) 3T3-L1 cells. B: Gene expression of late adipocyte markers adiponectin (Adipoq), leptin (Lep), peroxisome proliferator-activated receptor gamma (Pparg), and fatty acid-binding protein 4 (Fabp4) was analyzed by RT-qPCR using TaqMan technology. Open bars and open circles represent undifferentiated cells at day 0 prior to differentiation; gray bars, full circles correspond to mature adipocytes at day 11 after exposure of 3T3-L1 cells to the adipocyte differentiation medium 1 and 2 (n = 3). C: AGMO enzymatic activity in cell pellets of 3T3-L1 exposed to the basal medium at day 0 (open bars and open circles) and cells supplemented with the differentiation medium 1 and 2 at day 11 (gray bars and full circles) of adipogenesis (n = 3). D: Agmo gene expression of cells at day 0 (open bars and open circles) and day 11 (gray bars and full circles) of adipocyte differentiation. E: Time course of AGMO activity during differentiation of 3T3-L1 cells exposed to the standard hormonal differentiation cocktail (diff. med. control) or a hormonal induction medium devoid of either IBMX, RGZ, DEX, or IBMX/RGZ and DEX/RGZ in combination (n = 5). F: Lipid droplet quantification by Bodipy and Hoechst staining using the CellProfiler™ analysis software of 3T3-L1 cells at day 0 and day 11 incubated with the complete differentiation medium (control) or differentiation medium with omitted IBMX, RGZ, DEX, or IBMX/RGZ and DEX/RGZ (three areas per well; n = 4). Mean ± SEM except for the boxplots in F, which show the median ± interquartile range (IQR). Whiskers range from minimum to maximum. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, and ∗∗∗∗P < 0.0001.

Fig. 2

Agmo knockdown and the impact on lipid droplet mass and size during adipogenesis. A: AGMO enzymatic activity of 3T3-L1 cells harboring either a knockdown against the firefly luciferase as a control (black bars) or against Agmo (gray bars) at day 0 prior to differentiation and day 11 at the end of differentiation when mature adipocytes have formed (n = 5). B: Lipid droplet formation per cell of undifferentiated cells at day 0 and differentiated cells at day 11 using Bodipy for lipid droplet staining and Hoechst to stain cellular nuclei (four areas per well, n = 5). C: Violin plots showing lipid droplet size frequency distribution in the differentiated shLuc cell line (black) and the differentiated shAgmo knockdown cell line (gray) (12 areas per well, two wells per replicate, n = 3). The solid line represents the median and the staggered line the quartiles. D: Histogram showing the relative distribution of the lipid droplet size of shLuc adipocytes and shAgmo adipocytes (n = 3). Black bars and plots are representative for the shLuc control, while shAgmo cell lines are presented in gray. Mean ± SEM except for the boxplots in B, which show the median ± interquartile range (IQR). Whiskers range from minimum to maximum. ∗P < 0.05 and ∗∗P < 0.01.

Fig. 3

Remodeling of the cellular lipidome during adipocyte differentiation. A: Heatmap representation of the relative abundances of the 40 analyzed lipid classes for control (shLuc) and Agmo knockdown (shAgmo) after 0 and 11 days (d0 and d11). B: Pie chart of the relative abundances of the 40 major lipid classes during differentiation of the shLuc control and shAgmo from day 0 to day 11. All species are colored according to their respective lipid class indicated in the color legend on the right. C: Pie charts showing the relative abundance of neutral lipid species sorted according to their degree of desaturation in shLuc and shAgmo in vitro differentiated adipocytes at day 11. Total values below each chart indicate the cumulative relative abundance of all identified single lipid species that are shared in both cell lines and form the respective lipid class. Mean of N = 5 is shown.

Fig. 4

Detailed analysis of carbon chain length and saturation of single TG species upon Agmo knockdown in mature adipocytes. A: Volcano plot depicting the lipidomics data of TGs at day 11 of adipocyte differentiation. Significance cutoff is P < 0.01 as indicated by the horizontal dotted line. The vertical dotted line separates the log2(fold change) of decreased (blue dots) and increased (red dots) lipid species. B: Boxplots showing the relative abundances of TG at day 11 of adipocyte differentiation in the shLuc and shAgmo cell lines. A full list of accumulated or depleted metabolites is shown in supplemental Table S2.

Fig. 5

Behavior of lipid classes during 3T3-L1 adipocyte differentiation from day 0 to day 11 of adipocyte differentiation. Black symbols represent the shLuc cell line, and gray symbols depict the shAgmo cell line. Data are presented as mean ± SD, n = 5. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001.