Endocannabinoid activation at hepatic CB1 receptors stimulates fatty acid synthesis and contributes to diet-induced obesity

Abstract

Endogenous cannabinoids acting at CB(1) receptors stimulate appetite, and CB(1) antagonists show promise in the treatment of obesity. CB(1) (-/-) mice are resistant to diet-induced obesity even though their caloric intake is similar to that of wild-type mice, suggesting that endocannabinoids also regulate fat metabolism. Here, we investigated the possible role of endocannabinoids in the regulation of hepatic lipogenesis. Activation of CB(1) in mice increases the hepatic gene expression of the lipogenic transcription factor SREBP-1c and its targets acetyl-CoA carboxylase-1 and fatty acid synthase (FAS). Treatment with a CB(1) agonist also increases de novo fatty acid synthesis in the liver or in isolated hepatocytes, which express CB(1). High-fat diet increases hepatic levels of the endocannabinoid anandamide (arachidonoyl ethanolamide), CB(1) density, and basal rates of fatty acid synthesis, and the latter is reduced by CB(1) blockade. In the hypothalamus, where FAS inhibitors elicit anorexia, SREBP-1c and FAS expression are similarly affected by CB(1) ligands. We conclude that anandamide acting at hepatic CB(1) contributes to diet-induced obesity and that the FAS pathway may be a common molecular target for central appetitive and peripheral metabolic regulation.

Figure 1

Activation of CB₁ increases the gene expression of SREBP-1c, ACC1, and FAS in the liver of CB₁^+/+ mice. Mice were injected i.p. with vehicle, 20 ng/g HU210, 3 μg/g SR141716, or 20 ng/g HU210 plus 3 μg/g SR141716 (SR141716 + HU210) 1 hour prior to sacrifice and removal of the liver. RNA was isolated and Northern hybridization performed as described in Methods. An original blot (A) as well as the mean ± SEM from 5 replicate experiments in each group (B) are shown. Relative mRNA levels were quantified by densitometry, corrected for 18S ribosomal RNA levels used as loading control, and expressed as a percentage of the value measured in vehicle-treated controls.

Figure 2

Activation of CB₁ stimulates de novo fatty acid synthesis in the liver in vivo and in vitro. (A) In vivo fatty acid synthesis in CB₁^+/+ and CB₁^–/– mice on normal diet was assayed by the conversion of ³H₂O into fatty acids in the liver. Mice were treated i.p. with vehicle (white bars), 20 ng/g HU210 (black bars), 3 μg/g SR141716 (dark gray bars), or 20 ng/g HU210 plus 3 μg/g SR141716 (light gray bars), and the rate of fatty acid synthesis was expressed as micromoles of ³H₂0 converted into fatty acids per hour per gram of tissue weight. (B) In vitro fatty acid synthesis in isolated hepatocytes from mice. Isolated cells were treated with drugs in vitro and fatty acid synthesis quantified as described in Methods. Bars represent mean ± SEM from 4–5 separate experiments.

Figure 3

Presence of CB₁ in mouse liver. (A) CB₁ mRNA is present in the liver of CB₁^+/+ but not CB₁^–/– mice, as tested by RT-PCR. β-actin mRNA was amplified as internal control. (B) Localization of CB₁ mRNA in normal mouse liver by in situ hybridization in the presence (left panel) and absence (right panel) of the cRNA probe. (C) Immunoreactive CB₁ is present in hepatocytes in the liver of a CB₁^+/+ (left panel) but not a CB₁^–/– mouse (right panel). Center panel: the effect of preincubation of the N-terminal antibody with its blocking peptide in a section from the same liver as shown at left. Tissue structure is visualized by nuclear fast red counterstaining. (D) Immunoreactive CB₁ in purified liver plasma membranes was visualized by Western blot analysis using an antibody against the C terminus of rat CB₁. The specificity of the reaction is indicated by its absence in a preparation from a CB₁^–/– mouse. The expression of CB₁ is upregulated in mice on a high-fat diet (HF) compared to regular diet (R).

Figure 4

Effects of high-fat diet (black bars) versus regular chow (white bars) on physiological and biochemical parameters in CB₁^+/+ (n = 9 regular, 10 high-fat) and CB₁^–/– mice (n = 8 in each group). Food intake reflects cumulative intake over the diet period; the other parameters were measured at the time the mice were sacrificed. Adiposity index was calculated as the total fat pad weight ([subcutaneous + retroperitoneal + inguinal]/eviscerated body weight × 100). Body weight at the start of the diet was slightly, but significantly, lower in CB₁^–/– mice than in their CB₁^+/+ littermates, in agreement with published results (17). *P < 0.01 versus corresponding group on control diet; **P < 0.05 versus corresponding value in CB₁^+/+ mice.

Figure 5

High-fat diet induces fatty liver in CB₁^+/+ but not CB₁^–/– mice. Fat deposition is visualized by Oil Red O staining in liver sections from mice on normal diet (A and B) or on high-fat diet for 3 weeks (C and D) or for 14 weeks (E and F).

Figure 6

High-fat diet increases de novo hepatic fatty acid synthesis via activation of CB₁ receptors. The rate of conversion of ³H₂O to fatty acids in the liver was assayed in wild-type (A) and CB₁^–/– mice (B) 15 minutes after the i.p. injection of vehicle (white bars) or 3 mg/kg SR141716 (SR) (gray bars). Animals were on control or high-fat diets for 3 weeks prior to testing, as indicated. The marked increase in hepatic fatty acid synthesis in wild-type mice was inhibited in the presence of SR141716 and was absent in CB₁^–/– mice. *P < 0.01 versus corresponding vehicle-treated group. Bars indicate mean ± SEM from 5–8 animals in each group.

Figure 7

Cannabinoid regulation of lipogenic gene expression in the hypothalamus. (A) Activation of CB₁ by HU210 increases SREBP-1c and FAS gene expression in the hypothalamus of CB₁^+/+ mice (n = 4). The hypothalamus was dissected as previously described (39). RT-PCR analysis was performed as described in Methods, and its results were confirmed by real-time quantitative RT-PCR. (B) The CB₁ antagonist SR141716 inhibits SREBP-1c and FAS gene expression in fasted/refed (n = 6) but not in fasted-only mice (n = 6). Mice received an i.p. injection of vehicle (white bars) or 3 μg/g SR141716 (gray bars) at the end of the 24-hour fast.