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, 15 (3), 299-310

2-arachidonoylglycerol Signaling in Forebrain Regulates Systemic Energy Metabolism

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2-arachidonoylglycerol Signaling in Forebrain Regulates Systemic Energy Metabolism

Kwang-Mook Jung et al. Cell Metab.

Abstract

The endocannabinoid system plays a critical role in the control of energy homeostasis, but the identity and localization of the endocannabinoid signal involved remain unknown. In the present study, we developed transgenic mice that overexpress in forebrain neurons the presynaptic hydrolase, monoacylglycerol lipase (MGL), which deactivates the endocannabinoid 2-arachidonoyl-sn-glycerol (2-AG). MGL-overexpressing mice show a 50% decrease in forebrain 2-AG levels but no overt compensation in other endocannabinoid components. This biochemical abnormality is accompanied by a series of metabolic changes that include leanness, elevated energy cost of activity, and hypersensitivity to β(3)-adrenergic-stimulated thermogenesis, which is corrected by reinstating 2-AG activity at CB(1)-cannabinoid receptors. Additionally, the mutant mice are resistant to diet-induced obesity and express high levels of thermogenic proteins, such as uncoupling protein 1, in their brown adipose tissue. The results suggest that 2-AG signaling through CB(1) regulates the activity of forebrain neural circuits involved in the control of energy dissipation.

Figures

Figure 1
Figure 1. Biochemical characterization of MGL-Tg mice
(A) Schematic representation of the pMM403-CamKIIα-MGL-V5 construct utilized to generate MGL-Tg mice. The arrows indicate the position of the amplicon used for genotyping. (B) MGL mRNA levels in brain regions of wild-type mice (open bars) or MGL-Tg mice (closed bars). PFC: prefrontal cortex; Cx, rest of the cortex; Hipp, hippocampus; Str, striatum; Hypo, hypothalamus; Thal, thalamus; Cere, cerebellum. Results are expressed as mean ± SEM. **p<0.01 and ***p<0.001 (n=3). (C) MGL protein levels in brain regions of wild-type (Wt) or MGL-Tg mice. Arrows indicate the apparent molecular weight (MW, in kDa) of the MGL-V5 transgene, endogenous MGL and actin (used as standard). The experiment was repeated twice with identical results. (D) MGL activity and (E) 2-AG levels in various brain regions of wild-type (open bars) or MGL-Tg mice (closed bars). (F) anandamide (AEA) and arachidonic acid (AA) levels in the cortex. **p<0.01 and ***p<0.001 (n=6–7). (G) Levels of mRNAs encoding for CB1 (CB1R), diacylglycerol lipase-α (DGL-α), α-β-hydrolase domain 6 (ABHD6), N-acylphosphatidylethanolamine-specific phospholipase D (NAPE-PLD) and fatty acid amide hydrolase-1 (FAAH-1) in the forebrain of wild-type (open bars) or MGL-Tg mice (closed bars) (n=5). (H) MGL mRNA levels in various tissues of wild-type (open bars) or MGL-Tg mice (closed bars). Abbreviations: BAT, brown adipose tissue; WAT, white adipose tissue; LV, liver; GNG, sympathetic stellate ganglion. ***p<0.001 (n=3). The inset shows liver and GNG data in a magnified scale.
Figure 2
Figure 2. Neuroanatomical characterization of MGL-Tg mice
(A) Immunohistochemical localization of MGL in coronal brain sections from wild-type (Wt) and MGL-Tg (Tg) mice, using an antibody that recognizes both native and transgene-encoded MGL. The insets show nuclear staining with 4′,6-diamidino-2-phenylindole (DAPI). The dashed boxes marked b, d, and e highlight sections that are magnified in panel B–E. (B–C) Immunohistochemical localization of MGL in hippocampus of MGL-Tg mice. A laminar, punctuated immunostaining pattern (green) is observed in the stratum oriens (o), stratum radiatum (r), hilus (h) and stratum moleculare (m) of the dentate gyrus. Other abbreviations: p, stratum pyramidale; lm, stratum lacunosum-moleculare; g, stratum granulosum. DAPI staining is shown in blue. (D–E) Immunohistochemical localization of MGL in the paraventricular (PVN, D) and arcuate nuclei (ARC, E) of the hypothalamus in wild-type (Wt) and MGL-Tg (Tg) mice (dashed boxes d and e in panel A). The insets in D and E show negative control staining of adjacent slides using antibody pre-absorbed with purified MGL (0.18 mg/ml).
Figure 3
Figure 3. MGL-Tg mice are lean, hyperphagic and hypoactive
(A) Body-weight trajectory in wild-type (squares) and MGL-Tg mice (triangles) kept on a standard diet. *p<0.05 and ***p<0.001 (n=10). (B–C) Body adiposity (B) and lean mass (C) in 20 week-old wild-type (Wt, open bars) and MGL-Tg mice (closed bars), as assessed by magnetic resonance imaging. *p<0.05 and **p<0.01 (n=8). (D) Plasma triglycerides levels (mg/dl) in free-feeding (FF) or 4-h food-deprived (FD) wild-type (open bars) and MGL-Tg mice (closed bars). *p<0.05 and ***p<0.001 (n=3). (E) Serum glucose levels (mg/dl) in 4-h food-deprived wild-type (open bars) and MGL-Tg mice (closed bars). *p<0.05 (n=5). (F) Time-course (min) of glucose uptake (mg/dl) in wild-type (squares) and MGL-Tg mice (triangles). *p<0.05 and ***p<0.001 (n=5). (G) Water body content (% body mass) of 20 week-old wild-type (Wt, open bars) and MGL-Tg mice (closed bars), as assessed by MRI (n=8). (H) Meal frequency (number of meals/h) in adult wild-type (Wt, open bars) and MGL-Tg mice (closed bars). ***p<0.001 (n=10). (I) Time-course (h) of daily food intake in adult wild-type (squares) and MGL-Tg mice (triangles). *p<0.05 and ***p<0.001 (n=10). (J) Time-course (h) of daily motor activity (cumulative counts) in adult wild-type (squares) and MGL-Tg mice (triangles). **p<0.01 and ***p<0.001 (n=10 each).
Figure 4
Figure 4. MGL-Tg mice are resistant to high-fat diet-induced obesity
(A) Representative images of wild-type (Wt) and MGL-Tg mice (Tg) after 10 weeks of high-fat diet. (B) Body-weight trajectories (g) in wild-type mice (squares) and MGL-Tg mice (triangles) maintained on a high fat diet. *p<0.05 and ***p<0.001 (n=5–6). (C–E) Body weight gain (% initial body weight) (C), feed efficiency (g body weight/g food intake) (D), and fat mass (% body weight) (E) in wild-type (open bars) and MGL-Tg mice (closed bars) kept on a high fat diet. ***p<0.001 (n=5–6). (F) Triglyceride levels (μg/mg fresh tissue weight) in liver tissue from wild-type (open bars) and MGL-Tg mice (closed bars) kept on a high fat diet. ***p<0.001 (n=5–6). (G) Neutral lipids staining (Oil Red O) in sections of liver tissue from wild-type (Wt) and MGL-Tg mice (Tg) kept on a high fat diet. Magnification: top, 10x; bottom, 40x. (H–I) Plasma levels of insulin (H) and leptin (I) in wild-type (open bars) and MGL-Tg mice (closed bars) kept on normal (ND, n=3) or high-fat diet (HFD, n=5–6). *p<0.05 and ***p<0.001. (J–K) Plasma levels of glucose (J) and triglycerides (K) in wild-type (open bars) and MGL-Tg mice (closed bars) kept on a high-fat diet. **p<0.01 and ***p<0.001 (n=5–6)
Figure 5
Figure 5. MGL-Tg mice have high energy cost of activity and are hypersensitive by β3-adrenergic thermogenesis
(A) Regression analysis of energy expenditure (kcal/h/kg body weight) and motor activity (beam breaks) in wild-type (green circles) and MGL-Tg mice (red circles) kept on a normal diet. (B) Energy cost of motor activity (slope of regression curve in A) in wild-type (Wt, open bars) and MGL-Tg mice (Tg, closed bars). **p<0.01 (n=8). (C) Average body temperature in wild-type (Wt, open bars) and MGL-Tg mice (tg, closed bars). *p<0.05 (n=8). (D) Time-course (min) of body temperature changes elicited by β3-adrenergic agonist CL-316243 (0.1 mg/kg) in wild-type (squares) and MGL-Tg mice (triangles). CL-316243 was administered alone (left panel), together with MGL inhibitor JZL184 (16 mg/kg, 6 h before CL-316243) (center panel), or together with JZL184 plus CB1 inverse agonist rimonabant (10 mg/kg) (right panel). *p<0.05, **p<0.01 and ***p<0.001 (n=5–7). (E) Levels of mRNA encoding for β3-adrenergic receptor in BAT from wild-type (Wt, open bars) and MGL-Tg mice (Tg, closed bars) fed with normal chow. **p<0.01 (n=3). (F) Mitochondial complex activity (I + III) in BAT isolated from wild-type (Wt, open bars) and MGL-Tg mice (Tg, closed bars). *p<0.05, **p<0.01 and ***p<0.001 (n=3–4).
Figure 6
Figure 6. Increased mitochondria density in BAT of MGL-Tg mice
(A) Representative electron microscopy images of BAT from wild-type (Wt) and MGL-Tg mice (Tg). L, lipid vacuole; N, cell nucleus. (B) Ratio of mitochondria area to cytosol area in BAT from wild-type (Wt, open bars) and MGL-Tg mice (Tg, closed bars) fed with normal chow. *p<0.05 (n=3–4). (C–D). Levels of uncoupling protein-1 (UCP1) assessed by western blot analysis (C) or immunohistochemistry (D) in BAT from wild-type (Wt, open bars) and MGL-Tg mice (Tg, closed bars) fed with normal chow. Protein levels were quantified using the NIH Image J software and actin as a standard. **p<0.01 (n=4).
Figure 7
Figure 7. CART over-expression in the hypothalamus of MGL-Tg mice fed with normal chow
(A) CART mRNA levels in various brain regions of wild-type (Wt, open bars) and MGL-Tg mice (Tg, closed bars). ***p<0.001 (n=5–6). (B) Western blot analyses of CART in the hypothalamus of wild-type (Wt) and MGL-Tg (Tg) mice. The arrow indicates the apparent molecular weight of synthetic CART (55–102), which is shown in the blot on the right (Std). Note that synthetic CART migrates as a monomer, dimer or trimer (6, 12 and 18 kDa, respectively). Results are from one experiment replicated 4 times with similar results. (C) Localization of CART mRNA in the hypothalamus of wild-type (Wt) and MGL-Tg (Tg) mice. Abbreviations: ARC, arcuate nucleus; DMH, dorsomedial hypothalamus. (D) Immunohistochemical localization of CART peptide in the hypothalamus of wild-type (Wt) and MGL-Tg (Tg) mice. Abbreviation: PVN, paraventricular nucleus.

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