Triggering receptor expressed on myeloid cells 2 (TREM2) is known to be involved in the anti-inflammatory response and osteoclast development. However, the role of TREM2 in adipogenesis or obesity has not yet been defined. The effect of TREM2 on adipogenesis and obesity was investigated in TREM2 transgenic (TG) mice on a high-fat diet (HFD). To block TREM2 signaling, a neutralizing fusion protein specific for TREM2 (TREM2-Ig) was used. TG mice were much more obese than wild-type mice after feeding with an HFD, independent of the quantity of food intake. These HFD-fed TG mice manifested adipocyte hypertrophy, glucose and insulin resistance, and hepatic steatosis. The expression of adipogenic regulator genes, such as peroxisome proliferator–activated receptor γ and CCAAT/enhancer-binding protein α, was markedly increased in HFD-fed TG mice. Additionally, HFD-fed TG mice exhibited decreased Wnt10b expression and increased GSK-3β (glycogen synthase kinase-3β)–mediated β-catenin phosphorylation. In contrast, the blockade of TREM2 signaling using TREM2-Ig resulted in the inhibition of adipocyte differentiation in vitro and a reduction in body weight in vivo by downregulating the expression of adipogenic regulators. Our data demonstrate that TREM2 promotes adipogenesis and diet-induced obesity by upregulating adipogenic regulators in conjunction with inhibiting the Wnt10b/β-catenin signaling pathway.

Obesity is one of the major factors contributing to the development of metabolic syndromes, such as insulin resistance, type 2 diabetes (1), and hepatic steatosis (2). Obesity is characterized by an enlargement of the adipose tissue resulting from an increase in the number and/or size of the adipocytes, which is accompanied by chronic inflammation (2,3). These adipocytes secrete chemokines that recruit macrophages into the adipose tissue, leading to insulin resistance and inflammation due to the production of inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin (IL)-6, and IL-1β (2,3). Additionally, the enlarged adipose tissue releases excess free fatty acids (FFAs), which are taken up by hepatocytes and stored as triglycerides, resulting in hepatic steatosis and insulin resistance (2,4).

Adipogenesis is the process of differentiating mesenchymal stem cells into mature adipocytes (5). CCAAT/enhancer-binding proteins (C/EBPs) β and δ are key transcription factors in the early stage of adipogenesis as well as inducers of the expression of two principal adipogenic transcription factors, C/EBPα and peroxisome proliferator–activated receptor γ (PPARγ) (5). C/EBPα and PPARγ act synergistically to enhance the expression of adipogenesis-associated genes and subsequent adipogenic differentiation. The constitutive activation of Wnt/β-catenin pathway maintains the preadipocytes in an undifferentiated state by inhibiting the expression of C/EBPα and PPARγ (6,7). Consequently, canonical signaling through Wnt inhibits adipogenesis via the glycogen synthase kinase-3β (GSK3β)/β-catenin–dependent mechanism (68).

Triggering receptor expressed on myeloid cells 2 (TREM2) is expressed on the cell membrane of dendritic cells (9), macrophages (10), microglia (11,12), and osteoclasts (13) as an orphan cell surface receptor of the Ig superfamily. TREM2 associates with the adapter protein DAP12 containing an immunoreceptor tyrosine–based activation motif, which provides a docking site for the Syk (14). The recruitment and phosphorylation of Syk activate downstream intracellular signaling through phosphatidylinositol 3-kinase and extracellular signal–related kinase pathways (15). TREM2 signaling negatively regulates the inflammatory response via Toll-like receptor signaling in macrophages (14). In microglia, TREM2 signaling also inhibits the inflammatory response by suppressing TNF-α and nitric oxide synthase-2 genes and the phagocytic clearance of apoptotic neurons (12).

Recently, two independent groups have reported that TREM2 gene expression is gradually upregulated in the adipose tissue of dogs on a high-fat diet (HFD) (16) and in the mesenteric adipose tissue of insulin-resistant/diabetic db/db mice (17). However, the role of TREM2 in adipogenesis and obesity has been poorly defined. Moreover, the involvement of TREM2 in the Wnt signaling pathway has not yet been elucidated.

Here, we demonstrate for the first time that TREM2 induces obesity by promoting adipogenesis and upregulating the expression of adipogenic regulators. The current study supports the notion that the Wnt10b/β-catenin signaling pathway may play a crucial role in TREM2-induced adipogenesis and obesity.

Generation of TREM2 Transgenic Mice

Transgenic (TG) mice overexpressing TREM2 were generated by pronuclear microinjection, as described previously (18). The forward primer (5′-GAATTCGCCCTTGGCTGGCTGCTGGCA-3′) and reverse primer (5′-GTACGTGAGAGAATTC-3′) for mouse TREM2 were used to amplify TREM2 cDNA by RT-PCR. The PCR product was cloned into pGEM-T Vector (Promega, Madison, WI) and digested using EcoRI restriction enzyme, which was then cloned into pcDNA3.1 expression vector (Invitrogen, Carlsbad, CA). The pure isolated recombinant TREM2 gene was microinjected into a fertilized egg of a C57BL/6 mouse according to a standard protocol (Macrogen, Seoul, Republic of Korea). The integration of the transgene was confirmed by Southern blotting. Wild-type (WT) littermates and TG mice were maintained in a C57BL/6 genetic background, and any offspring was genotyped by RT-PCR prior to use in the experiments.

Mice, HFD, and Administration of TREM2-Ig

Eight-week-old male WT littermates and TG mice were fed with either standard diet (SD) or HFD D12451 (45 kcal% of energy from fat, 20 kcal% of energy from protein, and 35 kcal% of energy from carbohydrate) (Research Diets, New Brunswick, NJ) for 16 weeks under free-feeding conditions. Mice were individually housed and kept on a 12 h light/dark cycle at 22 ± 1°C. All of the mice experiments were conducted in accordance with the guidelines of the Institutional Animal Care Committee of Chonnam National University. The TREM2-Ig or control human Ig (hIg) was administered intraperitoneally twice per week for 16 weeks at 700 μg/mouse.

Cell Culture and Isolation of Primary Mouse Embryonic Fibroblasts

3T3-L1 mouse preadipocytes were obtained from the American Type Culture Collection (Manassas, VA). The primary mouse embryonic fibroblasts (MEFs) were isolated from embryos of WT and TG mice at 13.5 days postcoitus, as described previously (19).

Induction of Adipocyte Differentiation

MEFs and 3T3-L1 cells were cultured in DMEM supplemented with 10% bovine calf serum. Two days after reaching confluence (day 0), the medium was replaced with DMEM supplemented with 10% FBS containing DMI (0.25 ng dexamethasone, 250 μmol/L methylisobutylxanthine, 5 μg/mL insulin) (Sigma, St. Louis, MO). After 3 days, the medium was replaced with DMEM containing 10% FBS, dexamethasone, and insulin; and was replenished every other day for 10 days in 3T3-L1 cells or for 15 days in MEFs.

Indirect Calorimetry

Energy expenditure was measured by indirect calorimetry (Oxylet; Panlab, Cornellà, Spain) for 24 h after 16 weeks of SD or HFD, as described previously (20). Briefly, mice were acclimated individually in calorimetry chambers for 1 week prior to indirect calorimetry measurement. The values of VO2 and VCO2 were continuously monitored for 3 min at 30-min intervals. Food and water were freely available. Energy expenditure was expressed as VO2 (in milliliters per minute) per mouse with no normalization to body weight, and respiratory quotient (RQ) was calculated by dividing VCO2 by VO2.

Semiquantitative RT-PCR and quantitative real-time PCR

RT-PCR and quantitative real-time PCR were performed as described (4). The primers used for RT-PCR and quantitative real-time PCR are listed in Supplementary Table 1.

Western Blotting and Flow Cytometry Analysis

Western blotting was performed on cell lysates, as described previously (6). Cells were stained with the individual antibodies and analyzed by flow cytometry (BD Biosciences, San Diego, CA) (21). For intracellular staining, cells were fixed with 1.5% paraformaldehyde and stained with the individual antibodies after permeabilization with cold methanol.

Glucose and Insulin Tolerance Tests

After 16 weeks of feeding, mice fasted overnight were intraperitoneally injected with glucose (1 g/kg) or insulin (0.5 units/kg) for the glucose tolerance tests and insulin tolerance tests. Blood was collected from the tail vein at 0, 15, 30, 45, 60, and 75 min after injection. The blood glucose level was measured using a SureStep Plus Glucometer (LifeScan, Milpitas, CA).

Measurement of Plasma Metabolic Parameters

The metabolic parameters were determined using an Auto Chemistry Analyzer (Chiron, Emeryville, CA). The plasma levels of adiponectin, insulin, glucose, and leptin were determined using ELISA kits according to the manufacturer’s instructions (R&D Systems, Minneapolis, MN).

Histological Analysis and Oil Red O Staining

The liver and adipose tissues were embedded in frozen OCT compound and paraffin, respectively. The tissue sections were stained with hematoxylin-eosin (H&E) or Oil Red O (Sigma). For the Oil Red O staining of 3T3-L1 cells and MEFs, cells were fixed in 10% formalin, washed twice with 60% isopropanol, and stained with Oil Red O.

Statistical Analysis

The significance was calculated using two-way ANOVA and Student t test. The results were considered statistically significant when the P values were <0.05.

Generation of TREM2 TG Mice

A 732–base pair fragment of mouse TREM2 cDNA was inserted into EcoRI/EcoRI site of pcDNA3.1(+) expression vector containing pCMV promoter (Fig. 1A). To verify the expression of TREM2 in TG mice, TREM2 gene expression was analyzed by RT-PCR in various tissues. TG mice exhibited an overexpression of the TREM2 gene in all tissues examined in comparison with WT mice (Fig. 1B). TG mice also overexpressed TREM2 protein in epididymal white adipose tissue (EWAT), retroperitoneal white adipose tissue (RWAT), and liver tissue (Fig. 1C). The cell surface expression levels of TREM2 in the liver tissue of TG mice were also much higher than those in WT mice (Fig. 1D).

Figure 1

Generation and characterization of TREM2 TG mice. A: Schematic of the expression construct used to generate TREM2 TG mice. B: RT-PCR analysis of TREM2 gene expression in various tissues isolated from WT and TREM2 TG mice. C: Western blot analysis of TREM2 protein expression in the EWAT, RWAT, and liver tissue. D: Flow cytometry analysis of TREM2 expression on the liver cell surface. The data are representative of five independent experiments. bp, base pair.

Figure 1

Generation and characterization of TREM2 TG mice. A: Schematic of the expression construct used to generate TREM2 TG mice. B: RT-PCR analysis of TREM2 gene expression in various tissues isolated from WT and TREM2 TG mice. C: Western blot analysis of TREM2 protein expression in the EWAT, RWAT, and liver tissue. D: Flow cytometry analysis of TREM2 expression on the liver cell surface. The data are representative of five independent experiments. bp, base pair.

TREM2 TG Mice Are Much More Obese Than WT Mice After HFD Feeding

To elucidate the role of TREM2 in obesity, both WT and TG mice were fed with HFD. Whereas we observed no significant difference in body size between WT and TG mice, the bodies of TG mice were much fatter than those of WT mice on an HFD (Fig. 2A). The body weight of TG mice was considerably increased from 4 to 16 weeks after beginning the HFD feeding compared with WT controls (57.87 ± 1.20 g in TG vs. 46.48 ± 1.27 g in WT at 16 weeks) (Fig. 2B).

Figure 2

Increased body weight and adipocyte hypertrophy of HFD-fed TREM2 TG mice. Changes in the body size (A), body weight (B), and food intake (C) of WT and TG mice after 16 weeks of SD or HFD feeding (total n = 25/group). Representative images (D) and weights (E) of the EWAT, RWAT, and liver tissue isolated from WT and TG mice after 16 weeks of SD or HFD feeding (total n = 25/group). F: H&E staining of EWAT (a–d) and liver tissue (e–h) of WT and TG mice after 16 weeks of SD or HFD feeding and Oil Red O staining of the liver tissue (i–l). These images were captured under a light microscope at ×100 or ×200 magnification. The results are shown as the mean ± SEM and are representative of five independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 2

Increased body weight and adipocyte hypertrophy of HFD-fed TREM2 TG mice. Changes in the body size (A), body weight (B), and food intake (C) of WT and TG mice after 16 weeks of SD or HFD feeding (total n = 25/group). Representative images (D) and weights (E) of the EWAT, RWAT, and liver tissue isolated from WT and TG mice after 16 weeks of SD or HFD feeding (total n = 25/group). F: H&E staining of EWAT (a–d) and liver tissue (e–h) of WT and TG mice after 16 weeks of SD or HFD feeding and Oil Red O staining of the liver tissue (i–l). These images were captured under a light microscope at ×100 or ×200 magnification. The results are shown as the mean ± SEM and are representative of five independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001.

Next, we determined whether this difference in body weight was due to a disparity in food intake. No considerable difference was observed in food intake between WT and TG mice on an SD or an HFD, suggesting that the increased body weight of HFD-fed TG mice was not due to a difference in food intake (Fig. 2C). Consistently, the quantities of the EWAT and RWAT were markedly increased in HFD-fed TG mice, with comparable quantities in WT and TG mice on an SD (Fig. 2D). The liver tissue from TG mice was pale and enlarged compared with that in WT mice being fed an HFD (Fig. 2D), suggesting that hepatic steatosis may have occurred in TG mice. The weights of the EWAT, RWAT, and liver tissue were comparable between WT and TG mice being fed an SD but were greatly increased in HFD-fed TG mice (Fig. 2E). One of the classic rodent models of obesity is leptin-deficient ob/ob mice (22). Thus, to determine whether TREM2 was related to obesity, TREM2 gene expression was measured in ob/ob mice. Interestingly, TREM2 was markedly increased in the EWAT, RWAT, and liver tissue of ob/ob mice (Supplementary Fig. 1), implying that TREM2 may be involved in the development of obesity.

Histologically, although the adipocyte size in the EWAT was not different between WT and TG mice on an SD (Fig. 2F, a and b), the adipocytes from HFD-fed TG mice were considerably enlarged (Fig. 2F, c and d). The liver tissue size of HFD-fed TG mice paralleled that of the EWAT of the same mice (Fig. 2F, e–h). Additionally, a histochemical analysis using Oil Red O staining clearly indicated that the lipid droplets in the liver tissue of HFD-fed TG mice were larger than in those of WT mice (Fig. 2F, i–l).

TREM2 TG Mice Show Lower Energy Expenditure Than WT Mice on an HFD

Obesity is caused by excessive energy intake relative to expenditure (23). To determine whether changes in energy expenditure contribute to body weight gain in TG mice, energy expenditure was measured by indirect calorimetry. When energy expenditure was calculated without adjustment for body weight (24,25), nocturnal and diurnal energy expenditure was not significantly different between WT and TG mice on an HFD (Fig. 3A). Whereas nocturnal energy expenditure was significantly lower in SD-fed TG mice than in WT controls, diurnal energy expenditure was slightly lower. Nocturnal/diurnal RQ ratios were not significantly different between WT and TG mice on SDs and HFDs (Fig. 3B). Brown adipose tissue (BAT) has been known to play a crucial role in energy expenditure by promoting the expression of thermogenic genes, such as uncoupling protein 1 and β-3-adrenergic receptor (β-3AR) (26,27). While the expression of uncoupling protein 1 and β-3AR genes was decreased (Fig. 3C), the size and weight of BAT were remarkably increased in HFD-fed TG mice compared with WT controls (Fig. 3D and E).

Figure 3

Energy expenditure and thermogenesis-related gene expression in HFD-fed WT and TG mice. Energy expenditure (VO2; A) and RQ ratios (B) were measured by indirect calorimetry in WT and TG mice after 16 weeks of SD or HFD feeding. RQ was calculated by dividing VCO2 by VO2. Quantitative real-time PCR analysis for thermogenesis-related gene expression (C), representative images (D, ad) and H&E staining (D, eh), and weight (E) of BAT after 16 weeks of SD or HFD feeding. The data are represented as the mean ± SEM of three independent experiments. *P < 0.05; **P < 0.01.

Figure 3

Energy expenditure and thermogenesis-related gene expression in HFD-fed WT and TG mice. Energy expenditure (VO2; A) and RQ ratios (B) were measured by indirect calorimetry in WT and TG mice after 16 weeks of SD or HFD feeding. RQ was calculated by dividing VCO2 by VO2. Quantitative real-time PCR analysis for thermogenesis-related gene expression (C), representative images (D, ad) and H&E staining (D, eh), and weight (E) of BAT after 16 weeks of SD or HFD feeding. The data are represented as the mean ± SEM of three independent experiments. *P < 0.05; **P < 0.01.

TREM2 TG Mice Exhibit Severe Systemic Glucose and Insulin Resistance on HFD

The plasma adiponectin levels were decreased in HFD-fed TG mice compared with WT controls (Fig. 4A), but the leptin levels were elevated (Fig. 4B). However, food intake was not significantly different between the two groups of mice (Fig. 2C). These results are consistent with the previous findings that leptin is a critical regulator of obesity and appetite, but that plasma levels of leptin are not always correlated with food intake (28).

Figure 4

Systemic glucose tolerance and insulin resistance in HFD-fed TREM2 TG mice. The plasma levels of adiponectin (A), leptin (B), glucose (C), and insulin (D) were quantified by ELISA. Glucose (E) and insulin (F) tolerance tests were performed on the WT and TG mice after 16 weeks of SD or HFD feeding (n = 10). *P < 0.05; **P < 0.01.

Figure 4

Systemic glucose tolerance and insulin resistance in HFD-fed TREM2 TG mice. The plasma levels of adiponectin (A), leptin (B), glucose (C), and insulin (D) were quantified by ELISA. Glucose (E) and insulin (F) tolerance tests were performed on the WT and TG mice after 16 weeks of SD or HFD feeding (n = 10). *P < 0.05; **P < 0.01.

To determine whether the difference in adipose tissue mass leads to alteration of glucose metabolism mediated by TREM2 in mice on an HFD, we measured the plasma fasting glucose and insulin levels. In HFD-fed TG mice, the plasma levels of glucose and insulin were significantly higher than those in WT controls (Fig. 4C and D), suggesting that HFD-fed TG mice exhibited impaired glucose and insulin tolerance. We then performed glucose and insulin tolerance tests in mice on an SD or HFD. The plasma glucose levels did not significantly differ between WT and TG mice on an SD, but increased in TG mice on an HFD, indicating that HFD-fed TG mice were more glucose and insulin resistant than WT controls (Fig. 4E and F). The levels of insulin resistance–related genes, such as the insulin receptor, insulin receptor substrate 1/2, and glucose transporter 4, were reduced in the liver tissue and EWAT of HFD-fed TG mice (Supplementary Fig. 2A). Glucokinase (GK) gene expression was reduced in the liver tissue of HFD-fed WT and TG mice compared with those of SD-fed controls, which were more prominent in HFD-fed TG mice than in HFD-fed WT controls (Supplementary Fig. 2B). The phosphorylation levels of IRS1 at Ser302, AKT at Ser473, and Gsk3β at Ser9 were much lower in the EWAT, RWAT, and liver tissue of HFD-fed TG mice than in SD-fed TG mice (Supplementary Fig. 2C). Adipocyte-derived inflammatory chemokines, such as MCP-1 and chemokine ligand 5, recruit macrophages and T cells into the WAT, which leads to increased levels of inflammatory cytokines and thereby contributes to the induction of insulin resistance (29). The expression of IL-6, TNF-α, MCP-1, and chemokine ligand 5 was higher in the stromal vascular fraction (SVF) isolated from the EWAT of HFD-fed TG mice than in that of WT controls (Supplementary Fig. 3A). The percentages and absolute numbers of CD4+ and CD8+ T cells and F4/80+CD11c+ M1 macrophages were higher in the SVFs of HFD-fed TG mice than in those of WT controls (Supplementary Fig. 3B and C). However, the numbers of B220+ B cells and F4/80+CD206+ M2 macrophages were comparable between WT and TG mice on SDs and HFDs. These data indicate that TREM2 overexpression in mice on an HFD may play an important role in the development of insulin resistance.

TREM2 TG Mice Have Elevated Serum Metabolic Parameters Than WT Mice on an HFD

Elevated serum levels of cholesterol (CHOL), triglycerides, FFAs, and LDL are commonly observed in obesity (30,31). TG mice fed with an HFD manifested elevated serum levels of CHOL, triglycerides, LDL, and FFAs, whereas no significant differences between WT and TG mice fed with an SD were observed (Table 1). There were no significant changes in the plasma levels of glutamic oxaloacetic transaminase and glutamate-pyruvate transaminase in SD-fed WT and TG mice, but levels increased considerably in HFD-fed TG mice.

Table 1

TREM2 regulates plasma metabolic parameters and liver enzymes

SD
HFD
WT miceTG miceWT miceTG mice
CHOL (mg/L) 73 ± 6.4 79 ± 7.8 125 ± 11.3 243 ± 7.2* 
Triglycerides (mg/dL) 81 ± 3.0 88 ± 4.8 105 ± 2.0 119 ± 2.1 
LDL (mg/dL) 8 ± 1.4 10 ± 2.1 20 ± 5.7 67 ± 2.8* 
FFA (μEq/L) 1,154 ± 72.1 1,202 ± 32.5 1,342 ± 24.0 1,773 ± 46.7* 
GOT (IU/L) 95 ± 4.1 100 ± 3.4 128 ± 10.9 215 ± 8.8 
GPT (IU/L) 42 ± 2.1 50 ± 3.0 68 ± 4.0 99 ± 6.1 
SD
HFD
WT miceTG miceWT miceTG mice
CHOL (mg/L) 73 ± 6.4 79 ± 7.8 125 ± 11.3 243 ± 7.2* 
Triglycerides (mg/dL) 81 ± 3.0 88 ± 4.8 105 ± 2.0 119 ± 2.1 
LDL (mg/dL) 8 ± 1.4 10 ± 2.1 20 ± 5.7 67 ± 2.8* 
FFA (μEq/L) 1,154 ± 72.1 1,202 ± 32.5 1,342 ± 24.0 1,773 ± 46.7* 
GOT (IU/L) 95 ± 4.1 100 ± 3.4 128 ± 10.9 215 ± 8.8 
GPT (IU/L) 42 ± 2.1 50 ± 3.0 68 ± 4.0 99 ± 6.1 

All values correspond to the mean ± SEM of five experiments in each group (n = 5/group).

GOT, glutamic oxaloacetic transaminase; GPT, glutamate-pyruvate transaminase.

*P < 0.01;

P < 0.05.

Upregulation of Adipogenic Regulators and Inhibition of Wnt10b/β-Catenin Signaling in HFD-Fed TREM2 TG Mice

PPARγ and C/EBPα are regulators involved in promoting adipogenesis (5). Elevated levels of the lipogenic enzyme fatty acid synthase (FAS) are associated with obesity (32), and CD36 plays a role in the transport of fatty acids in adipocytes (33). We thus evaluated the expression of these adipogenic regulators in the EWAT of WT and TG mice on an SD or an HFD. Gene expression levels of PPARγ2 and C/EBPα were overexpressed in SD-fed TG mice compared with WT controls (Fig. 5A and B). Whereas the expression of PPARγ2, C/EBPα, FAS, and CD36 genes was markedly increased in HFD-fed TG mice compared with WT controls, the adiponectin levels were reduced, as determined by RT-PCR (A) and quantitative real-time PCR (B) (Fig. 5). Regarding PPARγ1 gene expression, previous reports have demonstrated that PPARγ2 transcript levels increase during adipogenic differentiation; in contrast, PPARγ1 is constitutively expressed (34). In agreement with these findings, we found that the expression of the PPARγ1 gene did not vary among the groups of mice studied here.

Figure 5

Elevated expression of adipogenic regulator genes and inhibition of Wnt10b/β-catenin signaling pathway in HFD-fed TREM2 TG mice. RT-PCR (A and C) and quantitative real-time PCR (B and D) analysis of the EWAT of WT and TG mice after 16 weeks of SD or HFD feeding. E: Western blot analysis of total and phosphorylated (p) GSK3β and β-catenin. The data in B and D are presented as relative fold changes, and the error bars represent the mean ± SEM of five independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 5

Elevated expression of adipogenic regulator genes and inhibition of Wnt10b/β-catenin signaling pathway in HFD-fed TREM2 TG mice. RT-PCR (A and C) and quantitative real-time PCR (B and D) analysis of the EWAT of WT and TG mice after 16 weeks of SD or HFD feeding. E: Western blot analysis of total and phosphorylated (p) GSK3β and β-catenin. The data in B and D are presented as relative fold changes, and the error bars represent the mean ± SEM of five independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001.

The activation of Wnt/β-catenin signaling pathway is required to inhibit adipogenesis (68). Wnt10b gene expression was dramatically reduced in the EWAT of TG mice on an SD and an HFD (Fig. 5), suggesting that TREM2 may regulate Wnt10b signaling. To test this hypothesis, we assessed the activation state of the TREM2 downstream signaling pathway. The phosphorylation of GSK3β at Ser9 was decreased in HFD-fed TG mice compared with WT mice (Fig. 5E), indicating that GSK3β was activated. In good accordance, GSK3β–mediated β-catenin phosphorylation at Ser33/37/Thr41 was increased in HFD-fed TG mice. These results imply that TREM2 on an HFD upregulated the expression of adipogenic regulators and inhibited Wnt10b/β-catenin signaling.

TREM2 Promotes Adipocyte Differentiation by Regulating Adipogenic Regulators

Differentiation of 3T3-L1 preadipocytes and MEFs into adipocytes was induced by DMI treatment. The degree of cell differentiation was determined based on the intracellular accumulation of lipid droplets, indicated by Oil Red O staining. The accumulation of cytoplasmic lipid droplets in 3T3-L1 cells was gradually enhanced during adipocyte differentiation (Fig. 6A).

Figure 6

Promotion of adipocyte differentiation through upregulation of adipogenic regulator gene expression by TREM2. Oil Red O staining (A), quantitative real-time PCR analysis (B), and RT-PCR analysis (C) of DMI-induced differentiation of 3T3-L1 preadipocytes into adipocytes. Oil Red O staining (D) and RT-PCR analysis (E) of DMI-induced differentiation of MEFs isolated from WT and TREM2 TG mice into adipocytes. The data in B are presented as relative fold changes, and the error bars represent the mean ± SEM of five independent experiments. F and H: Oil Red O staining of 3T3-L1 preadipocytes or MEFs pretreated with hIg or TREM2-Ig and cultured in the presence of DMI for 10 or 15 days, respectively. The hIg and TREM2-Ig were replenished every other day. G and I: RT-PCR analysis (left), and the relative expression ratio compared with hIg (right). The error bars represent the mean ± SEM of five independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 6

Promotion of adipocyte differentiation through upregulation of adipogenic regulator gene expression by TREM2. Oil Red O staining (A), quantitative real-time PCR analysis (B), and RT-PCR analysis (C) of DMI-induced differentiation of 3T3-L1 preadipocytes into adipocytes. Oil Red O staining (D) and RT-PCR analysis (E) of DMI-induced differentiation of MEFs isolated from WT and TREM2 TG mice into adipocytes. The data in B are presented as relative fold changes, and the error bars represent the mean ± SEM of five independent experiments. F and H: Oil Red O staining of 3T3-L1 preadipocytes or MEFs pretreated with hIg or TREM2-Ig and cultured in the presence of DMI for 10 or 15 days, respectively. The hIg and TREM2-Ig were replenished every other day. G and I: RT-PCR analysis (left), and the relative expression ratio compared with hIg (right). The error bars represent the mean ± SEM of five independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001.

Next, to investigate whether TREM2 is involved in adipocyte differentiation, we examined TREM2 gene expression during the differentiation process. TREM2 gene expression was increased in a differentiation stage–dependent manner (Fig. 6B). Similarly, the expression levels of adipogenic regulator genes, such as those encoding PPARγ1 and 2, C/EBPα, FAS, and CD36, also gradually elevated over time during differentiation (Fig. 6C). These data suggest that TREM2 may promote the differentiation of adipocytes through the regulation of adipogenic regulator expression. To evaluate this possibility, the degree of adipocyte differentiation and the expression of adipogenic regulator genes were evaluated in primary MEFs isolated from WT and TG mice. On days 10–15 of the differentiation period, the degree of intracellular lipid droplet accumulation in TG MEFs was much higher than in WT MEFs (Fig. 6D). Consistent with our data on the accumulation of lipid droplets, the gene expression levels of adipogenic regulators were markedly increased in TG MEFs on day 5 after adipogenic induction (Fig. 6E). These results indicate that TREM2 expression is increased during adipogenesis, which may accelerate the accumulation of lipid droplets by upregulating adipogenic regulators.

To confirm the involvement of TREM2 in adipocyte differentiation, the accumulation of lipid droplets and the expression of adipogenic regulator genes were evaluated following the blockade of TREM2 signaling by treatment with a neutralizing fusion protein specific for TREM2 (TREM2-Ig). The cytoplasmic lipid accumulation and droplet formation were substantially lower after treatment of 3T3-L1 cells with TREM2-Ig than with an hIg control (Fig. 6F). Additionally, the expression of the PPARγ2, C/EBPα, CD36, and FAS genes was suppressed in 3T3-L1 cells treated with TREM2-Ig (Fig. 6G). In contrast, Wnt10b gene expression was increased in TREM2-Ig–treated 3T3-L1 cells, suggesting that signaling via TREM2 may promote adipogenesis by downregulating Wnt10b expression. Consistent with the data in 3T3-L1 cells, intracellular lipid accumulation in primary MEFs was greatly decreased following TREM2-Ig treatment compared with hIg-treated controls (Fig. 6H). Furthermore, the treatment of primary MEFs with TREM2-Ig resulted in the suppression of PPARγ, C/EBPα, and FAS gene expression but elevated the expression of Wnt10b (Fig. 6I).

Blockade of TREM2 Signaling Inhibits HFD-Induced Obesity

TREM2-Ig decreased the magnitude of HFD-induced increase in the (final) body weight of both WT mice and TG mice, although the same phenomenon was not observed in hIg-administered mice (Fig. 7A–C). The TREM2-Ig–administered WT and TG mice also manifested a reduction in the sizes and weights of their EWAT and RWAT (Fig. 7D and E). However, the weight and size of the liver tissue was only reduced in TREM2-Ig–administered TG mice and not in WT controls. We also found that the HFD-induced increase in the expression of the PPARγ2, C/EBPα, FAS and CD36 genes was markedly inhibited in the TREM2-Ig–administered WT and TG mice compared with the hIg-administered control mice (Fig. 7F and G and Supplementary Fig. 4A). In contrast, adiponectin and Wnt10b gene expression was increased in both groups of TREM2-Ig–administered mice. In addition, the expression levels of insulin resistance–related genes were increased in the EWAT and liver tissue of TREM2-Ig–administered HFD-fed WT mice, suggesting that TREM2-Ig administration may improve adipose and hepatic insulin resistance (Supplementary Fig. 4B). Control hIg-administered HFD-fed TG mice showed a decreased phosphorylation of GSK3β compared with WT controls, but TREM2-Ig administration caused an increase in its phosphorylation (Fig. 7H). In contrast, the administration of HFD-fed TG mice with TREM2-Ig inhibited the increased phosphorylation of β-catenin in hIg-administered control mice.

Figure 7

Inhibition of HFD-induced obesity through the downregulation of adipogenic regulators by blockade of TREM2 signaling. Changes in the body size (A), body weight (B), and final body weight (C) of the hIg- or TREM2-Ig–administered WT and TG mice after 16 weeks of HFD feeding (total n = 25/group). The weights (D) and representative images (E) of the EWAT, RWAT, and liver tissue of these mice. RT-PCR (F), quantitative real-time PCR (G), and Western blot (H) analyses of the EWAT. The error bars represent the mean ± SEM of five independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 7

Inhibition of HFD-induced obesity through the downregulation of adipogenic regulators by blockade of TREM2 signaling. Changes in the body size (A), body weight (B), and final body weight (C) of the hIg- or TREM2-Ig–administered WT and TG mice after 16 weeks of HFD feeding (total n = 25/group). The weights (D) and representative images (E) of the EWAT, RWAT, and liver tissue of these mice. RT-PCR (F), quantitative real-time PCR (G), and Western blot (H) analyses of the EWAT. The error bars represent the mean ± SEM of five independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001.

Here, we provide the first demonstration that TREM2 promoted adipocyte differentiation through the regulation of adipogenic regulators in vitro. Furthermore, TREM2 TG mice showed the increased body weight, adipocyte hypertrophy, insulin resistance, and hepatic steatosis while being fed an HFD. In addition, HFD-fed TREM2 TG mice manifested reduced Wnt10b expression, suggesting that TREM2 may inhibit Wnt/β-catenin signaling pathway and promote diet-induced obesity. Therefore, TREM2 may act as a previously unrecognized inducer of obesity as well as a novel proadipogenesis factor.

The onset of obesity is strongly associated with age (35). The initial body weight of TG mice was lower than that of WT mice for up to 16 weeks after birth while being fed an SD (Fig. 2B). Despite the fact that TG mice weighed less at the onset of the experiment, after 12 weeks WT and TG mice weighted approximately the same. Thereafter, the body weight of TG mice gradually increased with age to a greater extent than that of WT mice. Although the increase in the body weight of TG mice appeared minimal, there was, in fact, a very large difference between WT and TG mice when the body weight changes were expressed as a percentage of the initial body weight (30% increase in WT mice and 61% increase in TG mice after 16 weeks of the experiment) (Supplementary Fig. 5). As shown in Fig. 2, no significant differences were found in the degree of obesity between young (up to 12 weeks old) WT and TG mice on an SD. However, aged (15 months old) SD-fed TG mice showed an increased body size and weight compared with WT controls (Supplementary Fig. 6A and B). A pronounced increase in the size and weight of the EWAT and liver tissue was also observed in SD-fed TG mice (Supplementary Fig. 6C and D). Histological analysis revealed that the adipocyte size of the EWAT from SD-fed TG mice was much larger than that from WT controls (Supplementary Fig. 6E). Gene expression of adipogenic regulators such as PPARγ2, C/EBPα, FAS, and CD36 was elevated, but the levels of adiponectin and Wnt10b were reduced in the EWAT of SD-fed TG mice (Supplementary Fig. 6F). Therefore, obesity also could be induced in TG mice by an HFD as well as by an SD, implying that TREM2 overexpression likely accelerates the development of age-related obesity, regardless of diet.

The expression of adipogenic regulators, such as PPARγ, C/EBPα, FAS, and CD36 was increased in HFD-fed TG mice, but obesity-induced insulin resistance was not improved. This may be partly due to impaired glucose and insulin metabolism by inhibiting GK and GLUT4 expression, insufficient binding sites for PPARγ and C/EBPα transcription factors by downregulating their target genes (GK, GLUT4), and FAS- or CD36-mediated inhibition of insulin signaling. Further investigations of the roles of these genes in obesity-induced insulin resistance from HFD-fed TG mice are required.

While TREM2 has been known primarily as a negative regulator of inflammatory response in macrophages, microglial cells, and dendritic cells (1114), a recent study (36) has reported that TREM2 amplifies the inflammatory response induced by Toll-like receptor in inflammatory bowel disease. However, whether TREM2 is involved in the inflammatory or anti-inflammatory response in adipose tissues has not yet been investigated. Here, HFD-fed TG mice revealed overexpression of chemokines, leading to recruitment of macrophages and T cells into adipose tissue. The elevated level of proinflammatory cytokines was also found in the SVFs of HFD-fed TG mice, which may be expressed by the infiltrated macrophages and T cells. The elevation of proinflammatory cytokines also contributes to the induction of insulin resistance (29). Our finding that insulin resistance was increased in HFD-fed TG mice is consistent with the finding that the infiltration of macrophages and T cells in adipose tissue plays a crucial role in the induction of insulin resistance by upregulating proinflammatory cytokines (29). Thus, our study elucidated the idea that TREM2 may be involved in the inflammatory response in adipose tissues.

In summary, our study is the first to demonstrate that TREM2 promotes adipogenesis by upregulating adipogenic regulators in conjunction with inhibiting the Wnt10b/β-catenin signaling pathway. Consequently, TREM2 is required for adipocyte differentiation, although the overexpression of TREM2 causes the development of obesity coupled with insulin resistance and hepatic steatosis. Our findings suggest that TREM2 acts as a novel marker and regulator of adipogenesis and that the blockade of TREM2 signaling is a potential therapeutic target in obesity and insulin resistance.

E.-M.K. is currently affiliated with the Department of Internal Medicine, University of Iowa and Veterans Affairs Medical Center, Iowa City, IA.

Funding. This work was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF), which was funded by the Ministry of Education, Science and Technology (grants NRF-2009-0077425 and NRF-2012R1A1A2004948).

Duality of Interest. No potential conflicts of interest relevant to this article were reported.

Author Contributions. M.P. designed the experiments, researched the data, contributed to the discussion, and wrote the manuscript. J.-W.Y. designed the experiments and researched the data. E.-M.K. contributed to the generation of the transgenic mouse model. I.-J.Y., S.-H.K., M.-G.S., and D.-H.K. researched the data. E.-H.L., H.-Y.L., K.-Y.J., K.-H.L., J.-H.J., and S.-S.O. contributed to the assist techniques and reagents. C.-H.Y. contributed to critical reading of the manuscript and useful discussions. K.-M.L. edited the manuscript. H.-S.K. designed the experiments; analyzed the data; and cowrote, reviewed, and edited the manuscript. H.-S.K. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

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Supplementary data