Beneficial Metabolic Effects of TREM2 in Obesity Are Uncoupled From Its Expression on Macrophages

Abstract

Obesity-induced white adipose tissue (WAT) hypertrophy is associated with elevated adipose tissue macrophage (ATM) content. Overexpression of the triggering receptor expressed on myeloid cells 2 (TREM2) reportedly increases adiposity, worsening health. Paradoxically, using insulin resistance, elevated fat mass, and hypercholesterolemia as hallmarks of unhealthy obesity, a recent report demonstrated that ATM-expressed TREM2 promoted health. Here, we identified that in mice, TREM2 deficiency aggravated diet-induced insulin resistance and hepatic steatosis independently of fat and cholesterol levels. Metabolomics linked TREM2 deficiency with elevated obesity-instigated serum ceramides that correlated with impaired insulin sensitivity. Remarkably, while inhibiting ceramide synthesis exerted no influences on TREM2-dependent ATM remodeling, inflammation, or lipid load, it restored insulin tolerance, reversing adipose hypertrophy and secondary hepatic steatosis of TREM2-deficient animals. Bone marrow transplantation experiments revealed unremarkable influences of immune cell-expressed TREM2 on health, instead demonstrating that WAT-intrinsic mechanisms impinging on sphingolipid metabolism dominate in the systemic protective effects of TREM2 on metabolic health.

Figure 1

Conserved visceral adipose upregulation of TREM2 in obesity. A: Trem2 expression in organs 13 weeks post-HFD. B: Trem2 expression in visceral and subcutaneous adipose depots 13 weeks post-HFD. C: Trem2 expression in ATMs and MA. D: Immunohistochemistry of eWAT depicting TREM2 13 weeks post-HFD. E: Trem2 expression in human visceral adipose biopsies obtained from individuals who were obIS and obIR. Bar graph bar data are mean ± SEM (n = 4 per diet). Statistical analysis was performed with Mann-Whitney U test (A and B) or two-way ANOVA followed by Bonferroni posttest (E). *P < 0.05, **P < 0.01, ****P < 0.0001. RP, retroperitoneal.

Figure 2

TREM2 deficiency aggravates obesity-induced insulin resistance. A: Insulin tolerance test (ITT) of WT and Trem2^–/– mice 13 weeks post-HFD (n = 8 per genotype). B: Area under the curve (AUC) of panel A. C: Oral glucose tolerance test (oGTT) of WT and Trem2^–/– mice 13 weeks post-HFD (n = 8 per genotype). D: Adipose weights 13 weeks post-HFD of animals in panel C. E: Mouse weights 13 weeks post-HFD of animals in panels C and D. F–H: Energy expenditure and activity of control and Trem2^–/– mice 13 weeks post-HFD (n = 4 per genotype). Data are mean ± SEM and are pooled for panels A–E from two independent experiments. Statistical analysis was performed with two-way ANOVA followed by Bonferroni posttest (A and C), Student t test (B and D), or one-way ANOVA followed by Tukey posttest (G). *P < 0.05, **P < 0.01. AU, arbitrary unit; RP, retroperitoneal; SAT, subcutaneous adipose tissue; VAT, visceral adipose tissue.

Figure 3

Elevated adipose hypertrophy of obese TREM2-deficient animals is associated with ATM remodeling. A: Representative hematoxylin-eosin staining of eWAT of WT and Trem2^–/– mice 13 weeks post-HFD (n = 8 mice per genotype). B: Quantification of adipocyte cell size of WT and Trem2^–/– mice 13 weeks post-HFD (n = 8 mice per genotype, and data correspond to size quantification of 3,492 WT and 2,103 Trem-2^–/– adipocytes). C: Absolute macrophage content (defined as viable CD45⁺F4/80⁺CD11b⁺ cells) in eWAT of both genotypes of animals following 13 weeks of feeding under both dietary conditions (n = 4 mice per condition). D: Percentage of macrophages (defined as viable CD45⁺F4/80⁺ cells) in eWAT of both genotypes of animals 13 weeks post-HFD (n = 4 mice per genotype). E: Ratio of FBC (viable CD45⁺F4/80⁺CD11b⁺CD11c⁺CD206^– cells) vs. FB206 (viable CD45⁺F4/80⁺CD11b⁺CD206⁺ cells) in eWAT of both genotypes of animals following 13 weeks of feeding under both dietary conditions (n = 4 mice per genotype). F: Percentage within the parent FB population of CD206⁺, CD11c⁺, and CD206⁺CD11c⁺ ATMs in both genotypes of obese animals 13 weeks post-HFD (n = 4 mice per genotype). G: Representative flow cytometry plots of panel F. Bar graph data are mean ± SEM and are pooled for panels A and B from two independent experiments. Data in panels E–G are representative of two independent experiments. Statistical analysis was performed with Student t test (B, D, and F) or one-way ANOVA followed by Tukey posttest (C and E). *P < 0.05, **P < 0.01, ****P < 0.0001. px², square pixels.

Figure 4

Metabolic stress is required for the protective effects of TREM2, which are linked to elevated sphingolipids levels. A: Log₂ fold change (FC) in metabolite abundance for Trem2^–/– over WT mice averaged over both dietary conditions compared with log₂(FC) metabolite levels of HFD over ND averaged over both genetic groups (n = 3 or 4 mice per genotype following 14 weeks of ND or HFD, respectively). B: Glucose levels (x-axis) vs. relative abundance of selected lipids (y-axis) following 45 and 60 min of insulin challenge. Dots represent mice, colored as indicated in the key. C: Activation of liver AKT signaling 5 min post–insulin injection in both genotypes fed an HFD for 13 weeks. D: Scheme for sphingolipid blockage. WT or Trem2^–/– mice were placed on an HFD for 13 or 26 weeks. Eight weeks post-DIO, mice were injected three times weekly with saline control or myriocin at a dose of 0.5 mg/kg and maintained on an HFD. E: Log₂FC in average abundance of short-, long-, and ultra-long-chain ceramides for both genotypes of myriocin-treated animals compared with saline controls (n = 4 mice per condition 26 weeks post-HFD). F: Insulin tolerance test (ITT) of both genotypes of mice in the context of sphingolipid blockage 13 weeks post-HFD (n = 8–9 mice per condition). G: Area under the curve (AUC) and mouse weights of data in panel F. H: Representative hematoxylin-eosin staining of eWAT 13 weeks post-HFD of animals in panels F and G (n = 8–9 mice per condition). I: Quantification of adipocyte cell size in panel H. Data are mean ± SEM. Statistical analysis was performed with two-way ANOVA followed by Bonferroni posttest (F) or one-way ANOVA followed by Tukey posttest (G and I). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. MW, molecular weight; oGTT, oral glucose tolerance test; px², square pixels.

Figure 5

Sphingolipid blockage exerts no effects on the ATM compartment of Trem2^–/– mice. A: Absolute eWAT macrophage content of both genotypes of animals (defined as viable CD45⁺F4/80⁺CD11b⁺ cells) in the context of sphingolipid blockage 13 weeks post-HFD (n = 5 mice per condition). B and C: Percentage within the parent FB population of CD206⁺ and CD11c⁺ ATMs in both genotypes of obese animals 13 weeks post HFD in the context of sphingolipid blockage (n = 5 mice per condition). D: ATM-mediated polarization and inflammation from flow-assisted cell-sorted viable CD45⁺CD3^-F4/80⁺CD11b⁺ cells of both genotypes of animals in the context of sphingolipid blockage 13 weeks post-HFD (n = 3–5 mice per condition). Data are fold change relative to WT saline animals. E: Percentage of macrophages (defined as viable CD45⁺F4/80⁺CD11b⁺ cells) in eWAT of both genotypes of animals in the context of sphingolipid blockage 13 weeks post-HFD (n = 3–5 mice per condition). F: Percentage of BODIPY⁺ macrophages (defined as viable BODIPY⁺CD45⁺F4/80⁺CD11b⁺ cells) in eWAT of both genotypes of animals in the context of sphingolipid blockage 13 weeks post-HFD (n = 3–5 mice per condition). Data are mean ± SEM. Statistical analysis was performed with one-way ANOVA followed by Tukey posttest (A–C, E, and F). *P < 0.05, ****P < 0.0001.

Figure 6

TREM2 exerts protective effects in sphingolipid-mediated secondary liver steatosis associated with specific defects in visceral ATM content. A: Insulin tolerance test (ITT) 26 weeks post-HFD (n = 4 per genotype). B: Liver size 26 weeks post-HFD (n = 4 per genotype). C: Liver steatosis 26 weeks post-HFD (n = 4 per genotype). D: Ratio of FBC (viable CD45⁺F4/80⁺CD11b⁺CD11c⁺ cells) vs. FB206 (viable CD45⁺F4/80⁺CD11b⁺CD206⁺ cells) in eWAT of both genotypes of animals 26 weeks post-HFD (n = 4 mice per genotype). E: Percentage of macrophages (viable CD45⁺Ly6G^–F4/80⁺) in eWAT 26 weeks post-HFD (n = 4 per genotype). F: Representative flow cytometry plots of panel E. G: Percentage of white blood cells (WBCs) evaluated using flow cytometry 26 weeks post-HFD (n = 4 per genotype). H: MCP-1 levels and protein content in ACM derived from WT and Trem2^–/– mice 26 weeks post-HFD (n = 5 mice per genotype). I: Migration levels of BM macrophages induced by ACM in panel H (n = 4 per condition). J: Representative liver morphology and hematoxylin-eosin staining 26 weeks post-HFD in the context of sphingolipid blockage (n = 4–5 mice per genotype). K: Liver steatosis 26 weeks post-HFD in the context of sphingolipid blockage (n = 4–5 mice per genotype). Data are mean ± SEM. Data in panels A–F, H, and I are representative of two independent experiments. Statistical analysis was performed with two-way ANOVA followed by Bonferroni posttest (A), Student t test (C–E and H), or one-way ANOVA followed by Tukey posttest (I and K). *P < 0.05, **P < 0.01, ****P < 0.0001. untr., untreated.

Figure 7

Uncoupling of effects of hematopoietic-expressed TREM2 on adipose hypertrophy and metabolic health. A: Scheme for BM transplantation studies. WT or Trem2^–/– mice were lethally irradiated and transplanted with either WT or Trem2^–/– BM to generate four groups of mice: WT>WT, Trem2^–/–>Trem2^–/– or Trem2^–/–>WT, and WT>Trem2^–/–. Posttransplant mice were maintained on an ND for 6 weeks, following which DIO was instigated. B: Insulin tolerance test (ITT) 13 weeks post-HFD. C: Area under the curve (AUC) of data in panel B. D: Oral glucose tolerance test (oGTT) 13 weeks post-HFD. E: AUC of data in panel D. F: Representative hematoxylin-eosin (H&E), TREM2, and F4/80 staining of eWAT of BM-transplanted mice 26 weeks post-HFD. G: Quantification of adipocyte cell size in H&E staining from panel F. Data correspond to size quantification of 3,868, 3,147, 3,198, and 4,412 adipocytes in WT>WT, Trem2^–/–>WT, Trem2^–/–>Trem2^–/–, and WT>Trem2^–/– mice, respectively. Data are mean ± SEM and are pooled from two independent experiments (n = 10–13 mice per genotype). Statistical analysis was performed with two-way ANOVA followed by Bonferroni posttest (B and D) or one-way ANOVA followed by Tukey posttest (C, E, and G). *P < 0.05, **P < 0.01, ***P < 0.01, ****P < 0.0001. AU, arbitrary unit; px², square pixels.

Figure 8

Effects of TREM2 on hepatic steatosis are linked to nonhematopoietic tissue and elevated adipose ceramide levels. A: Liver aspects and stainings of representative liver sections (hematoxylin-eosin [H&E], oil red O, ceramide) and representative whole mount of eWAT of BM-transplanted mice 26 weeks post-HFD. B: Liver steatosis in groups of BM-transplanted mice 26 weeks post-HFD. C: Serum levels of ALT 26 weeks post-HFD. D: Average abundance of short-, long-, and ultra-long-chain ceramide species in eWAT of BM-transplanted mice 26 weeks post-HFD. Individual ceramide abundances scaled between 0 (minimum) and 1 (maximum) (n = 4 mice per genotype). E: Serum adiponectin levels 26 weeks post-HFD. Data are mean ± SEM (B, C, and E) and pooled from two independent experiments (n = 10–13 mice per genotype). Statistical analysis was performed with one-way ANOVA followed by Tukey posttest (B, C, and E). *P < 0.05, **P < 0.01, ***P < 0.01.