Alterations in the hippocampal endocannabinoid system in diet-induced obese mice

Abstract

The endocannabinoid (eCB) system plays central roles in the regulation of food intake and energy expenditure. Its alteration in activity contributes to the development and maintenance of obesity. Stimulation of the cannabinoid receptor type 1 (CB(1) receptor) increases feeding, enhances reward aspects of eating, and promotes lipogenesis, whereas its blockade decreases appetite, sustains weight loss, increases insulin sensitivity, and alleviates dysregulation of lipid metabolism. The hypothesis has been put forward that the eCB system is overactive in obesity. Hippocampal circuits are not directly involved in the neuronal control of food intake and appetite, but they play important roles in hedonic aspects of eating. We investigated the possibility whether or not diet-induced obesity (DIO) alters the functioning of the hippocampal eCB system. We found that levels of the two eCBs, 2-arachidonoyl glycerol (2-AG) and anandamide, were increased in the hippocampus from DIO mice, with a concomitant increase of the 2-AG synthesizing enzyme diacylglycerol lipase-alpha and increased CB(1) receptor immunoreactivity in CA1 and CA3 regions, whereas CB(1) receptor agonist-induced [(35)S]GTPgammaS binding was unchanged. eCB-mediated synaptic plasticity was changed in the CA1 region, as depolarization-induced suppression of inhibition and long-term depression of inhibitory synapses were enhanced. Functionality of CB(1) receptors in GABAergic neurons was furthermore revealed, as mice specifically lacking CB(1) receptors on this neuronal population were partly resistant to DIO. Our results show that DIO-induced changes in the eCB system affect not only tissues directly involved in the metabolic regulation but also brain regions mediating hedonic aspects of eating and influencing cognitive processes.

Figure 1.

A, Growth curve of wild-type (CB₁^+/+) and CB₁ knock-out (CB₁^−/−) mice during 12 weeks of SD and HFD treatment. Data are expressed as mean ± SEM. CB₁^−/−; CB₁^+/+, n = 10 mice per group; GABA-CB₁^−/−, n = 7 mice. *p < 0.05, CB₁^+/+ SD vs CB₁^+/+ HFD; ^#p < 0.05, CB₁^+/+ SD vs CB₁^−/− SD; ⁺p < 0.05, CB₁^+/+ SD vs CB₁^−/− HFD; ^¥p < 0.001, CB₁^+/+ HFD vs GABA-CB₁^−/− HFD; two-way ANOVA analysis, Bonferroni's post hoc test. B, Total amount of visceral fat (in grams) in CB₁^+/+, CB₁^−/− mice, and GABA-CB₁^−/− under SD and HFD, respectively, measured at the end of the treatment. ***p < 0.001, CB₁^+/+ SD vs CB₁^+/+ HFD; **p < 0.01, CB₁^+/+ HFD vs GABA-CB₁^−/− HFD; t test analysis.

Figure 2.

2-AG and anandamide levels in different brain regions. A, B, Increase of 2-AG and anandamide in hippocampus of HFD-fed mice compared with SD-fed mice (n = 4, SD group; n = 8 mice, HFD group; t test analysis, *p < 0.05, **p < 0.01). C–F, No differences were observed in BLA and cerebellum. G, H, In the prefrontal cortex of mice fed with HFD, the levels of anandamide were slightly reduced (t test analysis, p < 0.01). Data are expressed in picomoles/milligram wet tissue ± SEM.

Figure 3.

A, CB₁ receptor immunoreactivity in hippocampi of SD-fed and HFD-fed animals (green signal: CB₁ receptor). B, Quantification of CB₁ receptor immunoreactivity in the area of the stratum radiatum, as indicated in A. C, CB₁ receptor quantification in the layer of pyramidal cell bodies as indicated in A. Signal was quantified in CA1, CA3, and dentate gyrus (DG). Scale bar, 250 μm. Data are expressed in integrated density ± SEM. n = 3 mice per group. Two-way ANOVA analysis, Bonferroni's post hoc test: **p < 0.01, ***p < 0.001.

Figure 4.

Stimulation of [³⁵S]GTPγS binding in hippocampal homogenates of HFD-fed (filled circles) and SD-fed (open circles) mice by various concentrations of the cannabinoid receptor agonist HU-210. Assays were performed in the presence of GDP (30 μm) and [³⁵S]GTPγS (0.05 nm) and were incubated for 60 min at 30°C as described in Materials and Methods. Nonspecific binding was determined in the presence of unlabeled GTPγS (30 μm). Basal binding was measured in the absence of receptor agonist and defined as 0% in each experiment. Data are expressed as percentage stimulation above basal [³⁵S]GTPγS binding and are the means ± SEM, n = 2, all performed in quadruplicate. EC₅₀ = 2.29 ± 0.04 nm (SD), 2.45 ± 0.06 nm (HFD); E_max = 68.83 ± 0.90 (SD), 68.74 ± 1.40 (HFD). Unpaired t test analysis, two-tailed: p = 0.9802.

Figure 5.

Western blot analysis of hippocampus homogenates of SD-fed and HFD-fed mice. A, DAGL-α protein levels were significantly increased in HFD-fed mice. B, DAGL-β protein levels were unchanged. C, D, Protein levels of MAGL and FAAH, two major eCB degradation enzymes, were unchanged. N = 3; data are normalized for α-tubulin. *p < 0.05, t test analysis. Error bars indicate SEM.

Figure 6.

A, DSI in CA1 hippocampal pyramidal neurons of HFD- and age-matched SD-fed mice. Bottom panel, Normalized eIPSCs amplitude with 3 s depolarization steps (HFD-fed mice: n = 12, filled circles; SD-fed mice: n = 11, open circles). Top panel, Representative traces of eIPSCs before (1) and after (2) DSI induction. B, Summary of DSI magnitude of SD- and HFD-fed wild-type mice, and CB₁^−/− SD-fed mice. Data are mean ± SEM. **p < 0.001, ***p < 0.01 versus respective baseline before depolarization (100%, dotted line). C, Effect of the inhibitor of 2-AG degradation JZL184, 100 nm on DSI. Bottom panel, Summary of DSI magnitude in SD-fed and HFD-fed mice before and after application of JZL184. Data are mean ± SEM. *p < 0.05, **p < 0.01 versus SD-fed mice without JZL184 treatment. Top panel, Representative traces of eIPSCs, 3 s after depolarization in untreated (1) and JZL184-treated (2) slices. D, Application of the CB₁ agonist WIN-55,212-2 (WIN) (5 μm) strongly reduced GABAergic currents with no significant difference between two diet groups.

Figure 7.

HFS induces a stronger I-LTD at CA1 pyramidal cells of HFD-fed mice compared with SD-fed mice. A, Summary of I-LTD induced in HFD-fed mice (n = 12; filled circles) compared with SD-fed mice (n = 5; open circles). B, Top panel, Representative traces of eIPSCs before (1) and after (2) I-LTD induction. Bottom panel, Summary of I-LTD amplitudes obtained by calculating the averaged responses 25–30 min after HFS with the last 5 min of baseline-averaged and normalized responses (**p < 0.01; ***p < 0.001). Data are mean ± SEM. C, Failure ratio in eliciting I-LTD between HFD-fed (filled bars) and SD-fed mice (open bars; n = 12 per group).