DGAT enzymes are required for triacylglycerol synthesis and lipid droplets in adipocytes

Abstract

The total contribution of the acyl CoA:diacylglycerol acyltransferase (DGAT) enzymes, DGAT1 and DGAT2, to mammalian triacylglycerol (TG) synthesis has not been determined. Similarly, whether DGAT enzymes are required for lipid droplet (LD) formation is unknown. In this study, we examined the requirement for DGAT enzymes in TG synthesis and LDs in differentiated adipocytes with genetic deletions of DGAT1 and DGAT2. Adipocytes with a single deletion of either enzyme were capable of TG synthesis and LD formation. In contrast, adipocytes with deletions of both DGATs were severely lacking in TG and did not have LDs, indicating that DGAT1 and DGAT2 account for nearly all TG synthesis in adipocytes and appear to be required for LD formation during adipogenesis. DGAT enzymes were not absolutely required for LD formation in mammalian cells, however; macrophages deficient in both DGAT enzymes were able to form LDs when incubated with cholesterol-rich lipoproteins. Although adipocytes lacking both DGATs had no TG or LDs, they were fully differentiated by multiple criteria. Our findings show that DGAT1 and DGAT2 account for the vast majority of TG synthesis in mice, and DGAT function is required for LDs in adipocytes, but not in all cell types.

Fig. 1.

Lack of lipid accumulation in MEFs deficient in both DGAT1 and DGAT2. A: The Kennedy pathway of TG synthesis. FA CoA, fatty acyl CoA; GPAT, glycerol-phosphate acyltransferase; AGPAT, acylglycerolphosphate acyltransferase; PAP, phosphatidic acid phosphatase. B: Single deletions of DGAT1 (D1KO) or DGAT2 (D2KO) do not affect adipogenesis as assessed by lipid accumulation (Oil-Red-O staining). UNDIFF, undifferentiated; DIFF, differentiated. C: Differentiation of MEFs deficient in both DGATs (D1D2KO) results in cells that lack lipid staining (top) and appear to lack LDs (bottom, phase-contrast microscopy image). Scale bar, 20 μm. Experiment was performed six times, with representative results shown. Exogenous FAs were not added during differentiation.

Fig. 2.

MEFs deficient in both DGATs (D1D2KO) have greatly reduced TG synthesis. A: DGAT activity is nearly absent in D1D2KO differentiated cells. Shown is an autoradiograph of radiolabeled lipids, separated by TLC, from in vitro DGAT assays. SE, sterol esters; TG, triacylglycerol; OA, oleic acid. B: Lack of TG synthesis in intact D1D2KO differentiated cells. Cells were incubated with [¹⁴C]oleic acid, and lipids were analyzed by TLC and autoradiography. C: Increased incorporation of labeled oleic acid into PC in intact D1D2KO differentiated cells. NL, neutral lipids; PE, phosphatidylethanolamine; PS, phosphatidylserine; PC, phosphatidylcholine; WT, wild-type. For B and C, cells were differentiated without exogenous FA and then loaded with 250 μM exogenous oleic acid during labeling. Experiments were performed three times, with representative results shown.

Fig. 3.

D1D2KO adipocytes have reduced TG levels. A: Charred TLC plate of lipid extracts from WT and D1D2KO differentiated cells. B: Quantification of mass spectrometry analysis of lipids from WT and D1D2KO adipocytes showing content of TG, free fatty acid (FFA), phosphatidylglycerol (PG), phosphatidylcholine, (PC), lysophosphatidylcholine (LPC). * P < 0.05, n = 3. Exogenous FAs were not added during differentiation. The experiment was performed three times, with mass spectrometry performed on a single sample set. Error bars represent SD.

Fig. 4.

D1D2KO newborn mice are severely depleted in TG. Lipid extracts from carcass, liver, and skin of newborn WT, D2KO, and D1D2KO mice were analyzed by TLC. The experiment was performed three times, with representative results shown.

Fig. 5.

D1D2KO adipocytes express perilipin but lack LDs and accumulate membranous aggregates. A: BODIPY493/503 (BODIPY, green) and perilipin (red) staining of WT and D1D2KO differentiated cells. Scale bar, 5 μm. B: WT and D1D2KO adipocytes visualized by electron microscopy. Lipid droplets are indicated by arrows. Mitochondria are indicated by black arrowheads. A large membranous aggregate is visible (outlined by white arrowheads) in the D1D2KO adipocyte. Aggregates were visible in all D1D2KO adipocytes. Scale bar, 1 μm. C: Western blot of perilipin in undifferentiated (DIFF–) and differentiated (DIFF+) WT (D1D2+) and D1D2KO (D1D2–) cells. A and B indicate the different isoforms of perilipin.

Fig. 6.

D1D2KO macrophages can make SE-containing LDs and can synthesize TG. A: WT and D1D2KO macrophages untreated (Control) or treated with oleic acid (500 μM) or acetylated LDL (25 μg/ml). Scale bar, 5 μm. Blue, Hoechst 33342; green, BODIPY 493/503. B: Analysis of lipids in conditions for A by TLC. C: TLC of WT and D1D2KO acetylated LDL-treated macrophages in the presence or absence of Sandoz 58-035 that have been incubated with [¹⁴C]oleic acid tracer without other exogenous oleic acid. The experiment was performed three times, with representative results shown. DAG, diacylglycerol.

Fig. 7.

D1D2KO differentiated cells have the cellular physiology of adipocytes. A: Results of microarray analyses of mRNA from WT and D1D2KO cells. Left: Venn diagram of genes regulated differently during differentiation in WT and D1D2KO adipocytes. Right: Scatterplot of induction values (differentiated/undifferentiated) for individual genes in the microarray. Points are color-coded based on meeting criteria for induction with differentiation (2-fold change and P < 0.001): red, meets criteria for both WT and D1D2KO; green, meets criteria for D1D2KO only; blue, meets criteria for WT only; gray, meets criteria for neither. Individual positions of three highly induced adipocyte markers (Adipoq, Fabp4, and Retn) are indicated. B: Expression of HSL, ATGL, and CGI-58 in WT (D1D2+) and D1D2KO (D1D2–) undifferentiated (DIFF–) and differentiated (DIFF+) MEFs. C: Intact phosphorylation of HSL at serine 563 (S563) in D1D2KO adipocytes induced by dibutyryl cAMP (db-cAMP). D: TG hydrolase activity in lysates of D1D2KO undifferentiated and differentiated MEFs and 3T3-L1 adipocytes. E: Glycerol release from WT and D1D2KO adipocytes untreated or treated with db-cAMP. F: Intact total (white bar) and HMW adiponectin (black bar) secretion in WT and D1D2KO differentiated MEFs. (G) Basal and insulin-stimulated glucose transport in WT and D1D2KO adipocytes. White bars, basal; black bars, insulin-stimulated. ND, none detected. * P < 0.05, n = 4. B–G, experiments were performed three times with representative results shown. Error bars represent SD.