2.2. dbh-CB1-KO Mice Show Reduced Weight, Adiposity, and Increased BAT Thermogenesis on a High-Fat Diet

HFD was reported to activate the SNS as well as the eCB system [26,38,39]. Therefore, we challenged both dbh-CB1-KO and -WT mice with HFD treatment. We found that dbh-CB1-KO mice gained less weight than dbh-CB1-WT in HFD conditions, despite the similar daily food intake (Figure 2A,B). Moreover, the dbh-CB1-KO mice on HFD showed an improved metabolic plasma profile, including reduced levels of plasma leptin (Figure 2C), insulin (Figure 2D), triglycerides, and low-density lipoprotein (LDL)/cholesterol (Figure 2E,F). Hepatic triglyceride accumulation was also decreased in KO compared with WT mice (Figure 2G).

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Figure 2

dbh-CB1-KO mice show a reduced body weight gain, increased thermogenic markers in BAT, decreased fat mass, and adipogenesis markers in visceral fat. (A) dbh-CB1-KO mice are resistant to HFD-induced obesity compared with WT (n = 15 each). *** p ≤ 0.001 by repeated measures two-way ANOVA. (B) dbh-CB1-KO mice show no differences in food intake (n = 15; Student t-test). (C) dbh-CB1-KO show decreased plasma leptin level (n = 8). ** p ≤ 0.01 by Student t-test. (D) Fasting plasma insulin levels in mice on HFD. (E) Lower plasma triglyceride levels in dbh-CB1-KO mice on HFD. (F) Lower LDL cholesterol levels in dbh-CB1-KO mice on HFD but no differences in HDL cholesterol levels compared with WT mice (n = 7–8). * p ≤ 0.05 by student t-test. (G) HFD-fed dbh-CB1-KO mice show significantly low liver triglyceride tissue levels (n = 4–5). * p ≤ 0.05 by student t-test. Data are expressed as mean ± SE. (H) dbh-CB1-KO show decreased visceral fat (n = 8–9). * p ≤ 0.05 by Student t-test. (I) Hematoxylin and eosin staining on histological sections from visceral fat tissue. Scale bar = 100 μm. (J) Quantitative analysis of adipocyte size (n = 3). ** p ≤ 0.01 by Student t-test. (K) dbh-CB1-KO show decreased Lep, Fas, and Cebpb mRNA levels in visceral fat whereas no significant differences are observed in the other markers of adipogenesis. (L) dbh-CB1-KO show increased Ucp1, Tfam, and Lipe in BAT. For (K,L) (n = 4), * p ≤ 0.05 by Student t-test. Data are expressed as mean ± SE.

Consistent with the lean phenotype and improved metabolic plasma profile, dbh-CB1-KO mice on HFD displayed less visceral fat and a reduced adipocyte size (Figure 2H–J). The gene expression analysis of markers in the visceral WAT showed reduced mRNA levels of leptin (Lep), fatty acid synthase (Fas), and CCAAT/enhancer-binding protein β (Cebpb) mRNA in dbh-CB1-KO mice compared with the control mice (Figure 2K). Gene expression analysis of thermogenic markers in BAT revealed increased mRNA levels of uncoupling protein (Ucp1), transcription factor A (Tfam) and Lipe (encoding hormone-sensitive lipase, Hsl) in dbh-CB1-KO mice compared to dbh-CB1-WT (Figure 2L).

2.3. dbh-CB1-KO Mice on a High-Fat Diet Show Increased Energy Expenditure and SNS Tone

Then, we analyzed the energy expenditure in these mice through indirect calorimetry. In agreement with the analysis of the thermogenic markers in BAT, the dbh-CB1-KO mice on HFD had increased oxygen consumption (Figure 3A), carbon dioxide production (Figure 3B), heat production (Figure 3C), and energy expenditure both during light and dark phases (Figure 3E,F). However, no differences in total ambulation (Figure 3D) were observed. ANCOVA analysis of the energy expenditure using body weight as a covariate showed significantly increased heat dissipation in dbh-CB1-KO mice compared with the control littermates (Figure 3G). In conclusion, HFD-fed dbh-CB1-KO mice showed reduced weight gain, predominantly caused by reduced adipogenesis in the visceral fat and a higher energy expenditure, most likely through enhancement of the β3-adrenergic signaling in the adipose tissue mediated by an increased SNS tone.

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Figure 3

dbh-CB1-KO mice show increased energy expenditure on HFD. (A) dbh-CB1-KO mice show increased oxygen consumption and (B) increased CO₂ release. (C) Heat production was increased although no significant statistical difference was observed in (D) ambulation in KO mice as compared with WT control (p ≤ 0.05 by two-way ANOVA; n = 5–8). dbh-CB1-KO mice showed high energy expenditure both in (E) light and (F) dark phase, and (G) ANCOVA analysis of the energy expenditure was calculated using body weight as the covariate. (H) Norepinephrine turnover (NETO) in the BAT of dbh-CB1-KO mice is increased after HFD treatment as compared with dbh-CB1-WT mice. (n = 6–9). * p ≤ 0.05. dbh-CB1-KO mice on HFD show increased β3-adrenergic receptor mRNA in BAT. For M, N, O, and P (n = 5–8), * p ≤ 0.05 by Student t-test. Data are expressed as mean ± SE.

In BAT, we analyzed the NE turnover, as a measure of the SNS tone, and β3 adrenergic receptor mRNA levels in these mice on HFD (Figure 3H). We observed that the dbh-CB1-KO displayed a significantly increased NE turnover (i.e., SNS tone) and enhanced β3 adrenergic receptor mRNA levels compared with the WT controls, which might be responsible for the leaner phenotype.

2.4. Lack of CB1 in dbh-Positive Neurons Enhances SNS Activity While Decreases NPY Activity in the Visceral Fat under Obesogenic Conditions

Next, we further detailed the NE and NPY activity in dbh-CB1-KO mice in obesogenic conditions. There is evidence that sympathetic neurons synthesize and co-secrete neuropeptides, such as NPY [40,41,42,43]. However, NPY exerts the opposite effect on adipose tissue expansion compared with NE. While NPY promotes the proliferation of adipocyte precursors via NPY type 1 receptor (NPY1r) [44], NE increases lipolysis and thermogenesis. To this end, we analyzed the changes in NE and NPY activity in HFD-fed mice (Figure 4). Dbh-CB1-KO mice showed reduced plasma NPY levels, and reduced Npy and Npy1r mRNA levels in visceral fat compared with the control littermates, suggesting decreased NPY signaling in the visceral fat (Figure 4A,B).

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Figure 4

HFD selectively enhances β-adrenergic signaling while decreases NPY signaling in the visceral fat of dbh-CB1-KO mice. (A) qPCR analysis of visceral fat from dbh-CB1-KO mice on HFD show decreased Npy and Npy1r gene expression (n = 5–6). (B) Lower plasma NPY levels in dbh-CB1-KO mice on HFD (n = 7). (C) dbh-CB1-KO mice on HFD show increased norepinephrine turnover in the visceral fat (n = 6–8). (D) dbh-CB1-KO mice on HFD show increased β1, β2, and β3 adrenergic receptor mRNA levels in the visceral fat (n = 6). (E,F) Representative blot and quantification analysis showing the several-fold increase in p-Hsl/Hsl ratio in the visceral fat of dbh-CB1-KO mice on HFD (n = 3–4). (G) dbh-CB1-KO mice show increased plasma NE levels in HFD conditions (n = 5–7). CB1 mRNA levels in (H) visceral fat and (I) BAT from dbh-CB1-KO and the corresponding control littermates. (J) Ucp1 mRNA in visceral fat. Data are expressed in mean ± SEM. Student t-test, * p < 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Further analysis of the visceral fat showed increased NE turnover as well as enhanced β1, β2, and β3 adrenergic receptor (β1AR, β2AR, and β3AR) mRNA levels, indicating enhanced NE signaling in the visceral fat (Figure 4C,D), thus protecting dbh-CB1-KO mice against HFD-induced obesity. A well-accepted readout of NE signaling is the phosphorylation of hormone-sensitive lipase [45,46,47]. Immunoblot analysis of visceral fat showed a significant increase in the phosphorylation of Hsl (p-Hsl/Hsl) in HFD-fed dbh-CB1-KO mice (Figure 4E,F). Additionally, dbh-CB1-KO showed higher plasma NE levels than the WT mice (Figure 4G). Recent studies have suggested that the activation of β3AR might lead to the upregulation of the eCB tone, thus limiting the increase in energy expenditure [48,49]. We found that CB1 and Ucp1 mRNA levels in the visceral fat of dbh-CB1-KO mice were not significantly different compared with the WT control littermates (Figure 4H,J). However, the CB1 mRNA expression was significantly increased in the BAT of dbh-CB1-KO mice compared with the WT control littermates (Figure 4I).

Taken together, CB1 deficiency in dbh-positive cells upregulated NE signaling and decreased NPY signaling in the visceral WAT upon HFD treatment compared with the WT mice, thus protecting dbh-CB1-KO mice against HFD-induced obesity.

4.2. Diet Experiments

Male mice, 7–8-weeks-old, singly housed, had free access to water and were fed ad libitum with a high-fat diet (HFD) for 3 months. In HFD, the caloric contribution was 60% fat, 17% protein, and 23% carbohydrate (Altromin, Lage, Germany; C1090-60). Body weight and food intake were recorded twice a week. At the end of the diet experiments, the mice were fasted and sacrificed by decapitation under isoflurane, and the trunk blood was collected in EDTA-coated tubes with a protease inhibitor, cooled on the ice, and immediately centrifuged at 1000× g for 5 min. The plasma was collected and kept frozen at −80 °C until it was analyzed for insulin, leptin, and lipids. The visceral fat was removed and weighed. Other tissues such as BAT, liver, brain, and sympathetic cervical ganglia were also removed and snap-frozen at −80 °C until analyzed. We used separate cohorts of mice for monitoring energy expenditure, NE turnover, and tissue collection. All of the experiments were carried out following the European council directive and were approved by the Ethical committee on animal care and use of Rhineland-Palatinate, Germany.

4.3. Plasma Hormone and Lipid Analyses

Plasma insulin and leptin were measured in overnight-fasted mice on HFD. Insulin was measured using ultrasensitive immunoassay kits (Alpco diagnostics, Salem, NH, USA; 80-INSMSU-E01). Leptin was measured using immunoassay kits from Millipore, Burlington, MA, USA (EZML-82K). The plasma total cholesterol and LDL cholesterol were measured using commercially available kits (BioVision, Waltham, MA, USA; K631-100). Plasma and hepatic triglycerides were measured by a triglyceride quantification kit (Bio Vision; K622-100). The plasma CORT was measured in mice (5–6-months-old) on HFD. Blood sampling was done within 30 s of mouse handling by rapid submandibular bleeding. The plasma CORT levels were measured following the instructions described in the kit (IBL International, Hamburg, Germany; RE52211).

The plasma NPY levels were measured in mice on HFD. Blood was quickly collected from the submandibular vein in EDTA vials with protease inhibitors within 30 s of mice handling. As platelets are a rich source of NPY, blood was centrifuged at 2000× g for 15 min at 4 °C to separate the plasma and platelets. The plasma was kept frozen at −80 °C until assayed. NPY in platelet-free plasma was measured by an enzyme-linked immunoassay kit (Millipore, Burlington, MA, USA; EZRMNPY-27K).

4.4. Indirect Calorimetry Measurements

Energy expenditure was measured by indirect calorimetry in the metabolic cages (TSE systems GmbH, Bad Homburg, Germany) six weeks after HFD treatment to monitor the metabolic activity over a 48-h period. Mice were acclimatized for 24 h in the cages before the start of the actual measurements. After the acclimatization, heat production, O₂ consumption, CO₂ production, locomotor activity, and food and water intake were measured every 15 min for the next 48 h. Energy expenditure was analyzed through an analysis of the covariance (ANCOVA) using body weight as a covariate in order to exclude the potential effect of body weight differences in the measurements [70].

4.5. Norepinephrine Turnover

NE turnover was determined in the adipose tissues of mice on HFD, through the administration of α-methyl-p-tyrosine (AMPT; Sigma, St. Louis, MO, USA; M8131), a competitive inhibitor of tyrosine hydroxylase, the rate-limiting enzyme in NE synthesis, as described earlier [71]. The mice from each genotype were divided into two groups, the first group was sacrificed, and adipose tissues were collected to measure basal level (time = 0). The other group was injected intraperitoneally with AMPT (0.25 mg/g BW), and the second dose of AMPT (0.125 mg/g BW) was injected 2 h after the first injection to maintain the inhibition of tyrosine hydroxylase. The mice were killed 4 h after the first AMPT injection and the adipose tissues were collected, snap-frozen in liquid nitrogen, and stored at −80 °C until assayed.

The NE tissue content was measured using an enzyme-linked immunoassay kit (LDN diagnostic, Nordhorn, Germany; BA E-5200). Briefly, ~100–150 mg of tissues were homogenized in a buffer containing 0.01 M HCl, 1 mM EDTA, and 4 mM sodium disulfite. After centrifugation for 10 min (10,000× g, 4 °C), NE was extracted from the supernatant following the instructions supplied with the ELISA kit. Finally, the NE turnover was calculated using the following formula: NE turnover = k[NE]₀, where k = (lg[NE]₀ − lg[NE]₄)/(0.434 × 4), [NE]₀ = basal NE concentration, and [NE]₄ = final NE concentration [72].

4.6. Histology and Immunohistochemistry of Adipose Tissue

The tissues were fixed in paraformaldehyde (4% in PBS), dehydrated in graded ethanol, cleared, and embedded in paraffin. Then, 10 µm thick sections were obtained and mounted on the slides. The slides were deparaffinized and stained with hematoxylin (Sigma, St. Louis, MO, USA; MHS16) and eosin (Sigma, St. Louis, MO, USA; 2853), as per the standard procedure. Sections were observed under a Leica DMRA microscope (Leica Microsystems, Wetzlar, Germany). For immunohistochemistry, the slides were deparaffinized and rehydrated with graded alcohol. After quenching the endogenous peroxidase, the tissue sections were blocked with blocking serum (VECTASTAIN Elite ABC Kit; Vector Laboratories, New York, NY, USA; PK-6102). Tissue sections were incubated with an anti-Ki-67 primary antibody (1:1000, Ki-67 mouse mAb; Cell Signaling, Danvers, MA, USA; 9449) overnight at 4 °C. The tissue sections were washed and detected using a secondary antibody from VECTASTAIN Elite ABC Kit (Vector Laboratories, New York, NY, USA; PK-6102). The images were captured with a Leica DMRA microscope (Leica Microsystems, Wetzlar, Germany) and the staining was quantified by ImageJ software (ImageJ version 1.45, NIH, Bethesda, MD, USA).

4.7. Real-Time qPCR

Tissue samples and brain punches were homogenized in 1 mL TRIzol (Invitrogen, Waltham, MA, USA; 15596-018). The total RNA was extracted and purified by RNeasy kit (Qiagen, Hilden, Germany; 74106) following the instructions supplied with the kit. cDNA was synthesized from 500 ng of total RNA using a high-capacity cDNA reverse transcription kit (Applied Biosystems, Waltham, MA, USA; 4390778). qPCR was performed using the Taqman gene expression master mix (Applied Biosystems, Waltham, MA, USA; 4369510) and the Taqman probes described in Table 1. All of the reactions were run in duplicate, and the expression levels of mRNA were calculated by the comparative Ct method normalized to the TATA-binding protein (Tbp). The mRNA expression levels in the neural tissues were normalized to glucuronidase beta (Gusb).

4.8. Immunoblot

The adipose tissue samples were homogenised in a RIPA buffer (50 mM Tris 8.0, 0.5% sodium deoxycholate, 0.1% SDS, 150 mM NaCl, 1% Triton X-100) containing protease and phosphatase inhibitors (Thermo Scientific, Waltham, MA, USA; 78445). Samples were cleared from lipids by using methanol and chloroform followed by TCA precipitation from the aqueous phase. For immunoblot, 2 μg of total protein was separated by SDS-PAGE and transferred to a nitrocellulose membrane. To detect p-Hsl, the membranes were blocked (5% milk in TBS-T buffer) and incubated overnight with rabbit anti-p-Hsl antibody (1:1000; Cell Signaling, Danvers, MA, USA; 4126) in the same blocking buffer. To detect the total Hsl nitrocellulose, the membranes were stripped with stripping buffer (2% SDS, 100 mM TCEP-HCl, 62.5 mM Tris 6.8) and the stripped membranes were again blocked (5% milk in TBS-T) and incubated with primary antibody against the total Hsl (1:1000; Cell Signaling, Danvers, MA, USA; 4107). The immunoreactivity was detected by peroxidase-conjugated anti-rabbit antibody and an enhanced chemiluminescence Western blot reagent kit (ECL, GE Healthcare, Amersham, UK). Glyceraldehyde 3-phosphate dehydrogenase (Gapdh) was used as loading control.

4.9. In Situ Hybridization

The mice were killed by decapitation under isoflurane and their brains were isolated, quickly frozen on dry ice, and stored at −70 °C until being used for sectioning. Sections of 20 µm thickness were cut on a cryostat. The DIG-labelled riboprobes for CB1 and dbh were prepared as described earlier [73]. The signal amplification was achieved by TSA plus system cyanine3/fluorescein (Perkin Elmer, NEL 744001KT, NEL 741001KT). The images were captured using a Leica DMRA microscope (Leica Microsystems, Wetzlar, Germany).

We are thankful to Michael Plenikowski for his contribution in drawing figures. We also thank Guiherme Horta and Konstantin Radyushkin for their contribution in the indirect calorimetry experiments.