Microarray analyses during adipogenesis: understanding the effects of Wnt signaling on adipogenesis and the roles of liver X receptor alpha in adipocyte metabolism

Abstract

Wnt signaling maintains preadipocytes in an undifferentiated state. When Wnt signaling is enforced, 3T3-L1 preadipocytes no longer undergo adipocyte conversion in response to adipogenic medium. Here we used microarray analyses to identify subsets of genes whose expression is aberrant when differentiation is blocked through enforced Wnt signaling. Furthermore, we used the microarray data to identify potentially important adipocyte genes and chose one of these, the liver X receptor alpha (LXR alpha), for further analyses. Our studies indicate that enforced Wnt signaling blunts the changes in gene expression that correspond to mitotic clonal expansion, suggesting that Wnt signaling inhibits adipogenesis in part through dysregulation of the cell cycle. Experiments designed to uncover the potential role of LXR alpha in adipogenesis revealed that this transcription factor, unlike CCAAT/enhancer binding protein alpha and peroxisome proliferator-activated receptor gamma, is not adipogenic but rather inhibits adipogenesis if inappropriately expressed and activated. However, LXR alpha has several important roles in adipocyte function. Our studies show that this nuclear receptor increases basal glucose uptake and glycogen synthesis in 3T3-L1 adipocytes. In addition, LXR alpha increases cholesterol synthesis and release of nonesterified fatty acids. Finally, treatment of mice with an LXR alpha agonist results in increased serum levels of glycerol and nonesterified fatty acids, consistent with increased lipolysis within adipose tissue. These findings demonstrate new metabolic roles for LXR alpha and increase our understanding of adipogenesis.

FIG. 1.

Experimental design. To investigate the preadipocyte and adipocyte phenotypes, RNA was purified from confluent 3T3-L1 preadipocytes (PA; day 0) and 3T3-L1 adipocytes that were 14 days postinduction (Ad; day 14). In addition, RNA was purified from mouse white adipose tissue (WAT) that had been separated into stromal vascular (SV) cells (which contain preadipocytes) and white adipocytes (WAd). To gain further insight into the early events of adipogenesis and the mechanism by which Wnt signaling blocks this differentiation process, 3T3-L1 cells were control infected (Control) or infected with a retrovirus encoding the gene for Wnt-1 (Wnt). Following selection, cells were induced to differentiate with methylisobutylxanthine, dexamethasone, and insulin (MDI) in 10% fetal calf serum. RNA was prepared from cells at 0, 16, 32, and 48 h following induction of differentiation. For each condition, RNA was purified from two independent samples, and transcript levels were measured by microarray with U74A Affymetrix chips, which represent approximately 10,000 distinct mouse genes and ESTs.

FIG. 2.

Genes (n = 119) that define the adipocyte phenotype. Criteria were established to identify genes that may be important in adipocyte function. These criteria were (i) transcripts that are expressed in 3T3-L1 adipocytes (Ad) and adipocytes from white adipose tissue (WAd) at similar levels (within twofold of one another) and (ii) transcripts that are highly expressed in both of these models relative to that observed in 3T3-L1 preadipocytes (PA) (at least fourfold higher and significantly different). Expression levels (10²) of these adipocyte genes are shown in panel A, and a partial list of these genes is given in panel B. The complete list of genes is available at http://www-personal.umich.edu/^∼macdouga/MacDougaldLab.html.

FIG.3.

Gene expression profiles during adipogenesis. RNA transcript levels were assessed from 3T3-L1 cells under six conditions: noninfected preadipocytes (PA), control-infected 3T3-L1 cells that had been induced to differentiate for 0, 16, 32, or 48 h, and day 14 adipocytes (Ad). A total of 1,889 genes that varied significantly in expression (P < 0.005) and by at least twofold in magnitude were selected for analysis by hierarchical clustering. Average expression for each gene was standardized to have mean = 0 and variance = 1 across conditions. Complete linkage clustering was performed with Cluster and Treeview software (http://www.microarrays.org/software.html). Genes are represented in rows, with a colorimetric scale (standard deviations [SDs] from the mean) to indicate relative expression levels; conditions are indicated above each column. Major patterns were defined as those nodes containing at least 150 transcripts and having a correlation coefficient of at least 0.85. Three quarters of the transcripts fell into one of five major patterns (A to E). Average expression profiles and SDs are shown for each pattern. This image, with a complete list of genes and their expression patterns, can be obtained at http://www-personal.umich.edu/^∼macdouga/MacDougaldLab.html.

FIG. 4.

Ectopic Wnt signaling alters mitotic clonal expansion. (A) Average expression profile and SD for 27 cell cycle genes are shown for control infected (Con) and Wnt infected (Wnt) cells. Average expression for each gene was standardized to have mean = 0 and variance = 1 across conditions. The full list of genes and their expression patterns can be obtained at http://www-personal.umich.edu/^∼macdouga/MacDougaldLab.html. (B) Control infected (Con) and Wnt infected (Wnt) cells were either lysed at time zero (0 h) or induced to differentiate and lysed 20 h later (20 h). Nuclei were purified, and nuclear lysates were separated by SDS-PAGE for immunoblot analysis of E2F4, p130, p27, and p21, as indicated. Two E2F4 isoforms are indicated (a and b). Similarly, p130 exists as two isoforms (a and c). Phosphorylation of the lower species (a) results in a mobility shift, resulting in b. These results are representative of two or three independent experiments.

FIG. 5.

(A) Genes within the early adipocyte phenotype are involved in energy storage. Of the genes and ESTs that cluster in the early adipocyte phenotype (Fig. 3D), 20 are involved in the storage of energy as glycogen, fatty acids, and triacylglycerol (TAG). Schematic diagram of the function of these genes in metabolism is shown to the left, with their names on the right. (B) Average expression profile and SD for C/EBPα, PPARγ, and SREBP1/ADD1, three transcription factors found within the early adipocyte phenotype cluster. Average expression for each gene was normalized to have mean = 0 and variance = 1 across conditions. TCA, trichloroacetic acid; OA, oxaloacetate; DHAP, dihydroxyacetone phosphate.

FIG. 6.

Genes within the late adipocyte phenotype are involved in energy mobilization Of the genes and ESTs that cluster in the late adipocyte phenotype (Fig. 3E), many are involved in energy mobilization. Schematic diagram of the function of these genes is shown, with members of cluster 3E highlighted in gray. β3AR, β3 adrenergic receptor; PKA, protein kinase A; HSL, hormone-sensitive lipase; PhK, phosphorylase kinase; PTG, protein targeted to glycogen; PP1, protein phosphatase 1.

FIG. 7.

Role of LXRα in adipocyte differentiation. (A) RNA from 3T3-L1 cells that had been induced to differentiate for the number of days indicated was analyzed by Northern for LXRα, SREBP1/ADD1, PPARγ, and C/EBPα. (B) 3T3-L1 preadipocytes were control infected (pBABE) or infected with a retrovirus encoding the gene for LXRα and selected with puromycin. Once confluent, cells were induced to differentiate in the absence (Con) or presence of the LXRα activator T0901317 (1 μM). Fourteen days later, cells were stained with Oil Red-O. These results are representative of three independent experiments.

FIG. 8.

Role of LXRα in adipocyte metabolism. Fully differentiated 3T3-L1 cells that expressed ectopic LXRα were incubated in the absence (Con) or presence of 1 μM T0901317 for 24 h. (A) Cells were labeled with [¹⁴C]glucose, and basal glucose uptake measurements were performed. (B) Lysates were analyzed by immunoblot for GLUT1. (C) Cells were labeled with [¹⁴C]acetate, and glycogen synthesis was measured. (D) NEFA and glycerol efflux from the cells into the medium was determined. (E) Cells were labeled with [¹⁴C]acetate, and lipids were extracted from the medium (top) or cell lysates (bottom) and separated by thin-layer chromatography. The origin, phospholipids (PL), cholesterol, diacylglycerol (DAG), and triacylglycerol (TAG) are indicated. These experiments are representative of at least three independent experiments.

FIG. 9.

Role of LXRα in lipid metabolism in vivo. Female C57BL/6 mice were injected with either vehicle (Con) or 50 mg of T0901317/kg/day daily for 1, 3, or 7 days, as described in Materials and Methods, and serum was analyzed for NEFA and glycerol. Three and seven days of T0901317 treatment resulted in significant (P < 0.01) changes in both NEFA and glycerol concentrations, as indicated (∗). Results are representative of two independent experiments.