ACAT1/SOAT1 maintains adipogenic ability in preadipocytes by regulating cholesterol homeostasis

Abstract

Maintaining cholesterol homeostasis is critical for preserving adipocyte function during the progression of obesity. Despite this, the regulatory role of cholesterol esterification in governing adipocyte expandability has been understudied. Acyl-coenzyme A (CoA):cholesterol acyltransferase/Sterol O-acyltransferase 1 (ACAT1/SOAT1) is the dominant enzyme to synthesize cholesteryl ester in most tissues. Our previous study demonstrated that knockdown of either ACAT1 or ACAT2 impaired adipogenesis. However, the underlying mechanism of how ACAT1 mediates adipogenesis remains unclear. Here, we reported that ACAT1 is the dominant isoform in white adipose tissue of both humans and mice, and knocking out ACAT1 reduced fat mass in mice. Furthermore, ACAT1-deficiency inhibited the early stage of adipogenesis via attenuating PPARγ pathway. Mechanistically, ACAT1 deficiency inhibited SREBP2-mediated cholesterol uptake and thus reduced intracellular and plasma membrane cholesterol levels during adipogenesis. Replenishing cholesterol could rescue adipogenic master gene-Pparγ's-transcription in ACAT1-deficient cells during adipogenesis. Finally, overexpression of catalytically functional ACAT1, not the catalytic-dead ACAT1, rescued cholesterol levels and efficiently rescued the transcription of PPARγ as well as the adipogenesis in ACAT1-deficient preadipocytes. In summary, our study revealed the indispensable role of ACAT1 in adipogenesis via regulating intracellular cholesterol homeostasis.

Keywords: PPARγ; adipocytes; cholesterol/metabolism; cholesterol/trafficking; cholesteryl ester; lipid rafts; nuclear receptors/SREBP.

Fig.1

ACAT1 is the dominant isoform in WAT from humans and mice. The relative mRNA levels of (A) ACAT1 and (B) ACAT2 in subcutaneous abdominal adipose tissue in the Lean-normal (n = 14) and Metabolic Healthy Obese (MHO; n = 25) subjects, based on RNA-seq data analysis. C: Acat1 and Acat2 mRNA levels in mature adipocytes isolated from epidydimal WAT (Epi-WAT) or inguinal WAT (Ing-WAT) of lean and high-fat diet-induced obese mice, respectively. D: ACAT1 and ACAT2 protein levels in Epi-WAT of lean, high-fat diet-induced DIO mice and ob/ob mice by western-blot (WB). E: SGBS preadipocytes were differentiated and collected at different time points during adipogenesis for the analysis of indicated genes using qPCR. White adipose stromal vascular fraction (SVF) differentiated adipocytes were collected at different time points during adipogenesis for the analysis of indicated genes using qPCR (F) and WB (G). Data is presented as Mean ± SEM (n = 3–5) and analyzed by student t test (A–C) or two-way ANOVA followed by Tukey’s test (E, F). ∗∗P < 0.01, ∗∗∗P < 0.001. Different letters indicate statistically significant difference (P < 0.05).

Fig. 2

ACAT1 deficiency in preadipocytes impairs adipogenesis. A: Schematic of knockout (KO) of ACAT1 in WAT-SVF with adenovirus (AV) and the subsequent adipogenesis. Ing-WAT derived SVF cells from Acat1^flox/flox mice were infected with CTRL-AV or CRE-AV (adenovirus expressing Cre recombinase) to generate either CTRL cells or ACAT1-KO cells, respectively. The ACAT1 protein levels were determined by Western blot (B). After differentiation, the adipocytes were stained with Oil Red O (C), and mRNA level of genes involved in adipogenic transcription program and TG synthesis were quantified by qPCR (D). ACAT1 was knocked down with lentivirus-mediated shRNA in human SGBS preadipocytes, which were further differentiated to mature adipocytes, and lipid droplets (LDs) were captured under a light microscope (E), and mRNA levels were quantified with qPCR (F). In 3T3-L1 cell line, shCTRL and shSOAT1 preadipocytes were collected to quantify for the mRNA levels of indicated genes with qPCR (G), and after differentiation, the LDs were stained with Nile red (H). Data is presented as Mean ± SEM (n = 3–5) and analyzed by student t test. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

Fig. 3

ACAT1 deficiency suppresses adipogenesis at the early stage. A: Schematic of cell samples for RNA-sequencing. B: in 3T3-L1 cell line, mRNA levels of adipogenic genes in shCTRL and shACAT1 adipocytes upon 3 days of differentiation were quantified by qPCR. C: top 10 downregulated signaling pathways analyzed by the biological process in 3T3-L1 differentiated shACAT1 versus shCTRL cells in Day 3. D: the expression of genes for cholesterol metabolism in 3T3-L1 shACAT1 versus shCTRL cells after 3 days’ adipogenic stimulation. E: the heatmap of genes with significant differences between shACAT1 versus shCTRL cells at Days 0, 3, and 6, respectively. F: the heatmap analysis of the key representative genes in the early stage of adipogenesis was shown to compare shACAT1 and shCTRL cells on Day 3. Data is presented as Mean ± SEM (n = 3–5) and analyzed by student t test. #P < 0.1, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

Fig. 4

Enhanced PPARγ pathway rescues adipogenesis in ACAT1 deficient cells. A: The heatmap analysis of the PPARγ targeted genes was performed in 3T3-L1 differentiated shACAT1 versus shCTRL cells after 3 days of differentiation. Rosiglitazone (2 μM) was added in shCTRL and shACAT1 (3T3-L1) cells at the early stage of adipogenesis (day 0–2), and mature adipocytes in each group were collected for quantifying (B) mRNA levels of adipogenic genes, as well as (C, D) lipid content with ORO. Oleic acid (0.5 mM) was supplemented in shCTRL and shACAT1 (3T3-L1) cells during the early stage of adipogenesis, mature adipocytes in each group were collected for quantifying (E, F) lipid content with ORO, and (G) mRNA levels of genes with qPCR. Data is presented as Mean ± SEM (n = 3–5) and analyzed by two-way ANOVA followed by Tukey’s test. Different letters indicate statistically significant difference (P < 0.05).

Fig. 5

ACAT1 deficiency attenuated Pparγ transcription by reducing plasma membrane (PM)-cholesterol. A: 3T3-L1 differentiated adipocytes were collected at different time points during adipogenesis for the analysis of cholesterol in total (TC), PM, and intracellular compartment, respectively. B: TC, PM and intracellular cholesterol levels were quantified in adipocytes differentiated from shCTRL and shSOAT1 3T3-L1 preadipocytes. C: cholesterol distribution in shCTRL and shACAT1 of 293T cells as indicated by RFP-domain 4, PM and nuclei as indicated by caveolin-1 (green) and nuclei (blue), respectively, were captured by confocal microscopy. The colocalization of D4 signal with caveolin-1 was quantified in the right panel. shCTRL and shACAT1 preadipocytes were differentiated to mature adipocytes. MβCD-coated cholesterol (20 μg/ml) were applied for 15 min to replenish cholesterol to PM, followed by cellular cholesterol level quantification with the biochemical kit (D), and mRNA analysis with qPCR (E). Data is presented as Mean ± SEM (n = 3–5) and analyzed by student t test (B, C) or two-way ANOVA (A, D, E) followed by Tukey’s test. #P < 0.1, ∗P < 0.05. Different letters indicate statistically significant difference (P < 0.05).

Fig. 6

ACAT1 deficiency impairs SREBP2-mediated cholesterol uptake. A: the heatmap analysis of the cholesterol homeostasis associated genes were performed in shACAT1 versus shCTRL adipocytes after 3 days of differentiation. B: mRNA levels of genes involved in cholesterol metabolism in adipocytes differentiated from control (CTRL-AV) and ACAT1 (CRE-AV) deficient preadipocytes. C: 3T3-L1 adipocytes differentiated from shCTRL and shACAT1 preadipocytes were cultured with 0.5 μg/ml 22-NBD-cholesterol and examined under confocal microscopy for quantifying CE associated green fluorescence intensity. The heatmaps of SREBP1 (D) and SREBP2 (E) targeted genes were performed in shACAT1 versus shCTRL adipocytes after 3 days of differentiation. F: shCTRL and shACAT1 3T3-L1 cells under the conditions of cholesterol depletion and repletion were collected for the quantification of n-SREBP2 and pre-SREBP2 with WB, followed with the quantifications of band intensity with ImageJ (G–I). J: luciferase activity in shCTRL and shACAT1 adipocytes (3T3-L1) which were transfected with the SRE-luciferase plasmid. Data is presented as Mean ± SEM (n = 3–5) and analyzed by student t test. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

Fig. 7

ACAT1 helps maintain adipogenic ability by mediating cholesterol homeostasis. A: Acat1 mRNA level in 3T3-L1 preadipocytes with ACAT1 or ACAT1-mutant overexpression. In shCTRL and shACAT1 preadipocytes, we overexpressed human catalytically functional ACAT1 and induced adipogenesis. Then the total cellular cholesterol level (B), lipid level with ORO staining (C, D), and mRNA level with qPCR (E) were quantified. shCTRL and shACAT1 preadipocytes were differentiated for 3–4 days and glucose uptake ability was measured with 2-DG6P in the absence (F) or the presence (G) of insulin. Overexpressing ACAT1 and catalytic-dead ACAT1-mutant in shACAT1 preadipocytes, and subsequently differentiate for 6 days. Lipid staining with ORO (H) and gene expressions with q-PCR (I) were quantified. (J) schematic illustration the absence of ACAT1 leads to impaired adipogenesis in adipocytes. Data is presented as Mean ± SEM (n = 3–5) and analyzed by two-way ANOVA followed by Tukey’s test. Different letters indicate statistically significant difference (P < 0.05).

Fig. 7

ACAT1 helps maintain adipogenic ability by mediating cholesterol homeostasis. A: Acat1 mRNA level in 3T3-L1 preadipocytes with ACAT1 or ACAT1-mutant overexpression. In shCTRL and shACAT1 preadipocytes, we overexpressed human catalytically functional ACAT1 and induced adipogenesis. Then the total cellular cholesterol level (B), lipid level with ORO staining (C, D), and mRNA level with qPCR (E) were quantified. shCTRL and shACAT1 preadipocytes were differentiated for 3–4 days and glucose uptake ability was measured with 2-DG6P in the absence (F) or the presence (G) of insulin. Overexpressing ACAT1 and catalytic-dead ACAT1-mutant in shACAT1 preadipocytes, and subsequently differentiate for 6 days. Lipid staining with ORO (H) and gene expressions with q-PCR (I) were quantified. (J) schematic illustration the absence of ACAT1 leads to impaired adipogenesis in adipocytes. Data is presented as Mean ± SEM (n = 3–5) and analyzed by two-way ANOVA followed by Tukey’s test. Different letters indicate statistically significant difference (P < 0.05).