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. 2017 Nov 1;127(11):4148-4162.
doi: 10.1172/JCI83626. Epub 2017 Oct 16.

Adipocyte Cannabinoid Receptor CB1 Regulates Energy Homeostasis and Alternatively Activated Macrophages

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Adipocyte Cannabinoid Receptor CB1 Regulates Energy Homeostasis and Alternatively Activated Macrophages

Inigo Ruiz de Azua et al. J Clin Invest. .
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Abstract

Dysregulated adipocyte physiology leads to imbalanced energy storage, obesity, and associated diseases, imposing a costly burden on current health care. Cannabinoid receptor type-1 (CB1) plays a crucial role in controlling energy metabolism through central and peripheral mechanisms. In this work, adipocyte-specific inducible deletion of the CB1 gene (Ati-CB1-KO) was sufficient to protect adult mice from diet-induced obesity and associated metabolic alterations and to reverse the phenotype in already obese mice. Compared with controls, Ati-CB1-KO mice showed decreased body weight, reduced total adiposity, improved insulin sensitivity, enhanced energy expenditure, and fat depot-specific cellular remodeling toward lowered energy storage capacity and browning of white adipocytes. These changes were associated with an increase in alternatively activated macrophages concomitant with enhanced sympathetic tone in adipose tissue. Remarkably, these alterations preceded the appearance of differences in body weight, highlighting the causal relation between the loss of CB1 and the triggering of metabolic reprogramming in adipose tissues. Finally, the lean phenotype of Ati-CB1-KO mice and the increase in alternatively activated macrophages in adipose tissue were also present at thermoneutral conditions. Our data provide compelling evidence for a crosstalk among adipocytes, immune cells, and the sympathetic nervous system (SNS), wherein CB1 plays a key regulatory role.

Conflict of interest statement

Conflict of interest: The authors have declared that no conflict of interest exists.

Figures

Figure 1
Figure 1. Adipocyte CB1 deletion prevents DIO and metabolic dysregulation.
(A) Body weight growth curves of Ati-CB1–KO (n = 21) and Ati-CB1–WT (n = 24) littermates on SD. Tamoxifen-induced recombination occurred for 5 days at 5 weeks of age (arrow). Tam, tamoxifen. (B) Body weight growth curves of Ati-CB1–WT (n = 22) and Ati-CB1–KO (n = 19) mice on HFD. Tam-induced recombination occurred for 5 days at 5 weeks of age (arrow). The switch from SD to HFD was at the beginning of week 7. (C) 3D visualization (ventral view) of skeleton (white) and total adipose tissue (red) from in vivo micro-CT images of Ati-CB1–WT and Ati-CB1–KO mice (19 weeks of age) on SD and HFD, respectively. (D) Quantification of total fat content (as percentage of total mouse volume) by in vivo micro-CT analysis in Ati-CB1–WT and Ati-CB1–KO mice on SD and HFD, respectively. (E) Comparison of body weight growth curves of Ati-CB1–KO (n = 19) mice and Total-CB1–KO (n = 8) mice on HFD. (FL) Plasma profiles of Ati-CB1–WT (n = 4–9) and Ati-CB1–KO (n = 4–8) on both diet treatments (at 19 weeks of age). Data are shown as mean ± SEM. *P < 0.05; #P < 0.01; P < 0.001. Two-way ANOVA (A, B, E); Student’s t test (D); 1-way ANOVA (FL).
Figure 2
Figure 2. CB1-dependent gene expression profile in adipocytes is depot specific.
(AF) Gene expression analysis (relative units) of Pparg, Cebpa, Lep, Fasn, Adipoq, and Acaca in EF, SF, and MF from Ati-CB1–WT and Ati-CB1–KO on SD (WT, n = 5–9; KO, n = 5–8), and HFD (WT, n = 5–8; KO, n = 5–6). (G) Representative images of EF, SF, and MF from Ati-CB1–WT and Ati-CB1–KO on both SD and HFD. H&E staining. Scale bars: 100 μm. (HK) Gene expression analysis (relative units) of Cox8b, Ucp1, Ppargc1a, and Adrb3 in SF from Ati-CB1–WT (n = 4–7) and Ati-CB1–KO (n = 4–7) on both SD and HFD. (L) Electron microscopy of SF from Ati-CB1–WT and Ati-CB1–KO on both SD and HFD. Scale bar: 1 μm. mito, mitochondrium; lp, lipid droplet. (MO) Gene expression analysis (relative units) of Cox4i2, Ucp1, and Adrb3 in BAT from Ati-CB1–WT (n = 6) and Ati-CB1–KO (n = 6–8) on both SD and HFD. Data are shown as mean ± SEM. *P < 0.05; #P < 0.01; P < 0.001 vs. WT, Student’s t test.
Figure 3
Figure 3. CB1 deletion in adipocytes affects caloric intake and EE and promotes alternative macrophage activation.
(A) Daily caloric intake (in kJ) of Ati-CB1–WT (SD fed, n = 11; HFD fed, n = 21) and Ati-CB1–KO mice (SD fed, n = 17; HFD fed, n = 27) on SD and HFD. (B) Pair-feeding experiment. Body weight curves of Ati-CB1–WT (n = 8) and Ati-CB1–KO (n = 13) on HFD. Tissue NE turnover in EF (C), SF (D), and BAT (E) from Ati-CB1–WT (n = 3) and Ati-CB1–KO mice (n = 4) on HFD. (F) EE in Ati-CB1–WT (n = 16) and Ati-CB1–KO mice (n = 13) on HFD. (G) Ambulatory activity during indirect calorimetry recording in Ati-CB1–WT (n = 16) and Ati-CB1–KO (n = 13) on HFD. (HJ) Gene expression analysis (relative units) of markers for alternatively activated macrophages (Mrc1, Clec10a) in EF, SF, and BAT from Ati-CB1–WT and Ati-CB1–KO mice on HFD (n = 11–12). (K) Protein markers for alternatively activated macrophages (CD206 and CD301) were monitored by flow cytometry in EF from Ati-CB1–WT and Ati-CB1–KO mice on HFD (n = 11–12). (L) Gene expression (relative units) of the catecholamine-synthesizing enzymes (Th, Dbh, Ddc) and alternatively activated macrophage markers (Mrc1, Clec10a) in CD11b+F4/80+-sorted ATMs from WAT of Ati-CB1–WT (n = 6–11) and Ati-CB1–KO (n = 5–11) mice on HFD. Dbh mRNA levels were measured by ddPCR analysis. (M) TH protein levels were measured by flow cytometry in CD11b+F4/80+-sorted ATMs (left) and CD301+ cells (M2 macrophages, right) in WAT from Ati-CB1–WT (n = 9) and Ati-CB1–KO (n = 9) mice on HFD. Data are shown as mean ± SEM. *P < 0.05; #P < 0.01; P < 0.001, Student’s t test (A, CE, HM); 2-way ANOVA (B, F, G).
Figure 4
Figure 4. CB1 deletion in adipocytes promotes alternatively activated macrophages and remodeling of adipose tissue preceding body weight changes.
(A) Body weight of 7-week-old SD-fed Ati-CB1–WT (n = 13) and Ati-CB1–KO mice (n = 10) 2 weeks after tamoxifen-induced CB1 deletion in adipocytes. (B) Relative CB1 mRNA levels are strongly decreased in EF, SF, and BAT from 7-week-old Ati-CB1–KO (n = 9–12) mice as compared with WT controls (n = 8–9) on SD. (C) Representative images of EF, SF, and BAT from Ati-CB1–WT and Ati-CB1–KO on SD. H&E staining. Scale bar: 100 μm. (D) Quantification of adipocyte cell size (arbitrary units) in EF from Ati-CB1–WT (n = 3) and Ati-CB1–KO (n = 3). (E) Gene expression analysis (relative units) in EF of markers of adipocyte differentiation (Pparg, Cebpa) and lipogenesis (Fasn, Acaca) and of adipokines (Lep, Adipoq) (n = 9). (F) Gene expression analysis (relative units) of alternatively activated macrophages (Mrc1, Clec10a) in EF (n = 9). (G) Protein analysis of markers for alternatively activated macrophages (CD206 and CD301) was monitored by flow cytometry in EF from Ati-CB1–WT (n = 13) and Ati-CB1–KO (n = 13) on SD. (H) Gene expression (relative units) of catecholamine-synthesizing enzymes (Th, Dbh, Ddc) in whole EF from Ati-CB1–WT (n = 3–8) and Ati-CB1–KO (n = 5–13) on SD. (I) Tissue NE levels in EF from Ati-CB1–WT (n = 8) and Ati-CB1–KO (n = 8) mice on SD. (J) Gene expression analysis (relative units) of thermogenic markers (Cox8b, Ucp1, Ppargc1a) in SF (n = 8–12). (K) Gene expression analysis (relative units) of thermogenic markers (Cox4i2, Ucp1) in BAT from Ati-CB1–WT (n = 8) and Ati-CB1–KO (n = 12) mice on SD. Data are shown as mean ± SEM. *P < 0.05; #P < 0.01; P < 0.001 vs. WT, Student’s t test.
Figure 5
Figure 5. Adipocyte-specific CB1 deletion in obese mice mediates weight loss and reverses obesity-related metabolic alterations.
(A) Body weight growth curves of Ati-CB1–WT–TAO (n = 8–10) and Ati-CB1–KO–TAO (n = 6–7) on both SD and SHFD before (weeks 4 to 16) and after (weeks 17 to 22) tamoxifen-induced CB1 deletion. (B) Body weight variation 6 weeks after tamoxifen-induced CB1 deletion in mice on SHFD. (C) Fasting blood glucose in Ati-CB1–WT–TAO (n = 8) and Ati-CB1–KO–TAO (n = 8) on SHFD. (DH) Plasma levels of insulin, leptin, adiponectin, PAI1, and IL-6 in Ati-CB1–WT–TAO (n = 4–9) and Ati-CB1–KO–TAO (n = 4–8) on SD and SHFD. (I) Glucose and insulin tolerance tests in Ati-CB1–WT–TAO (n = 9) and Ati-CB1–KO–TAO (n = 10) on SHFD. (J) Gene expression for markers of alternatively activated macrophages (Mrc1, Clec10a) in EF and SF from Ati-CB1–WT–TAO (n = 7) and Ati-CB1–KO–TAO (n = 5) on SHFD. (K) Chronic treatment with the peripherally acting CB1 antagonist AM6545 (10 mg/kg, i.p.) or its vehicle and analysis of body weight in Ati-CB1–WT–TAO (n = 8–7) and Ati-CB1–KO–TAO (n = 6) on SHFD. Data are shown as mean ± SEM. *P < 0.05; #P < 0.01; P < 0.001, 2-way ANOVA, Bonferroni’s post hoc test (A, K, I); Student’s t test (B, C, J); 1-way ANOVA (DH).

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