Peripheral cannabinoid-1 receptor inverse agonism reduces obesity by reversing leptin resistance

Abstract

Obesity-related leptin resistance manifests in loss of leptin's ability to reduce appetite and increase energy expenditure. Obesity is also associated with increased activity of the endocannabinoid system, and CB(1) receptor (CB(1)R) inverse agonists reduce body weight and the associated metabolic complications, although adverse neuropsychiatric effects halted their therapeutic development. Here we show that in mice with diet-induced obesity (DIO), the peripherally restricted CB(1)R inverse agonist JD5037 is equieffective with its brain-penetrant parent compound in reducing appetite, body weight, hepatic steatosis, and insulin resistance, even though it does not occupy central CB(1)R or induce related behaviors. Appetite and weight reduction by JD5037 are mediated by resensitizing DIO mice to endogenous leptin through reversing the hyperleptinemia by decreasing leptin expression and secretion by adipocytes and increasing leptin clearance via the kidney. Thus, inverse agonism at peripheral CB(1)R not only improves cardiometabolic risk in obesity but has antiobesity effects by reversing leptin resistance.

Figure 1. JD5037 Is a Peripherally Restricted CB₁ Receptor Inverse Agonist

(A) Structure of JD5037 and its brain-penetrant parent SLV319. (B) JD5037 has 20 times higher CB₁ binding affinity than SLV319 (left), and both are inverse agonists, as tested by GTPγS binding (right). Each curve represents the mean of three experiments performed in triplicate. (C) Brain and plasma levels of JD5037 and SLV319 1 hr following acute or 28 day oral dosing at 3 mg/kg, n = 4–7 animals. Both compounds accumulate in liver, with hepatic levels after acute versus chronic administration of 3,676 ± 37 versus 6,262 ± 540 ng/g (JD5037) and 817 ± 88 versus 4,384 ± 386 ng/g (SLV319). (D) Pretreatment of mice with SLV319, but not with JD5037 (3 mg/kg p.o. 1 hr prior to test), results in displacement of PET ligand from brain CB₁R. Scans from typical experiments are shown, with statistics from three to six replicates. *p < 0.05 relative to values in vehicle pretreated mice. (E) SLV319, but not JD5037 (3 mg/kg i.v. each), displaces bound CB₁ PET ligand. For crystallographic structure, physicochemical features, and behavioral profile of JD5037 see also Figure S1. Vertical bars represent SEM.

Figure 2. SLV319 and JD5037 Cause Similar Metabolic Effects in DIO Mice

(A) Transient reduction of daily food intake, n = 7 mice/group. (B) JD5037 does not induce taste aversion, as tested in lean mice. n = 6–7/group. *p < 0.001 relative to vehicle. ^#p < 0.01 relative to LiCl group. (C and D) (C) SLV319 and JD5037 (3 mg/kg/day for 28 days) normalize body weight and (D) reduce adiposity. n = 7/group, *p < 0.005 relative to STD, ^#p < 0.01 relative to HFD vehicle group. (E) JD5037 treatment reduces body fat without affecting lean body mass, as assessed by MRI. N = 6/group. * and ^# as in (D). (F–I) Reversal of HFD-induced steatosis (hepatic triglycerides) and hepatocellular damage (plasma ALT, AST). * and ^# as in (D). (J–M) Reversal of HFD-induced hyperglycemia, hyperinsulinemia, glucose intolerance (ipGTT), and insulin resistance (ipIST). * and ^# as in (D). (N and O) Reversal of HFD-induced hyperleptinemia and hypoadiponectinemia. * and ^# as in (D). (P and Q) Improved plasma lipid profile, * and ^# as in (D). For additional effects on substrate utilization and energy expenditure, see Figure S4. Vertical bars represent SEM.

Figure 3. Metabolic Effects of JD5037 in DIO, ob/ob, and db/db Mice

(A–C) JD5037 (3 mg/kg/day, 7 days) reduces body weight, adiposity, and food intake in DIO but not in ob/ob or db/db mice. *p < 0.05 relative to vehicle, n = 5–6 mice/group. (D and E) JD5037 treatment attenuates the hyperglycemia and hyperinsulinemia in DIO, ob/ob, and db/db mice. (F and G) JD5037 attenuates the glucose intolerance (ipGTT) and insulin resistance (ipIST) in DIO and ob/ob mice. *p < 0.5 relative to corresponding vehicle values. (H–J) JD5037 reduces hepatic triglycerides, plasma ALT, and AST in all three strains. (K) JD5037 attenuates hyperleptinemia in DIO and db/db mice, no plasma leptin in ob/ob mice. (L and M) JD5037 causes similar increases in plasma adiponectin and FFA in DIO, ob/ob, and db/db mice. For the lack of an acute hypophagic effect of JD5037 in a fasting/refeeding paradigm, see Figure S3, and for the lack of effect of JD5037 on daily food intake and body weight in CB₁R^−/− mice, see Figure S2. Vertical bars represent SEM.

Figure 4. JD5037 Causes Hypophagia and Weight Loss in DIO Mice via Endogenous Leptin

(A and B) Pretreatment of DIO mice with a leptin antagonist (GESLAN, 5 μg/g/day) prevents the weight loss and hypophagia caused by JD5037, 3mg/kg/day (treatment start at ↑). *p < 0.05 relative to GESLAN + vehicle group, n = 5 mice/group. (C and D) Reversal of HFD-induced hyperleptinemia by JD5037, 3 mg/kg/day, but not by pair feeding, precedes reversal of the weight gain. *p < 0.05 relative to corresponding vehicle values, n = 8/group. (E and F) Positive correlation between reversal of hyperleptinemia and reduction of either body weight or daily food intake, as calculated from data in (B)–(D). Vertical bars represent SEM.

Figure 5. Inverse Agonism at Peripheral CB₁R Reverses Leptin Resistance

(A) Effect of leptin (3 mg/kg twice daily for 4 days) on body weight in lean and DIO mice and in DIO mice treated for 7 days with JD5037, 3 mg/kg/day p.o. n = 4/group, *p < 0.05 relative to corresponding vehicle-treated group. (B) Effect of leptin on food intake in the same groups as in (A). (C) Leptin-induced phosphorylation of hypothalamic STAT3 is suppressed in DIO mice, but not in DIO mice pretreated with JD5037 or SLV379, 3/mg/kg/day for 28 days, as visualized by western blotting. Means ± SE from six mice per group were analyzed by densitometry. (D and E) HFD-induced increase in hypothalamic SOCS3 mRNA and protein is reversed by chronic JD5037 treatment. (F) Plasma leptin, food intake, and body weight change in DIO mice chronically infused with saline, 1 or 3 μg/g/day mouse leptin, or 3 μg/g/day human leptin, and treated for 7 days with vehicle or JD5037, 3 mg/kg/day. *p < 0.05 relative to vehicle. (G) JD5037 increases basal pSTAT3 and restores leptin-induced pSTAT3 in DIO mice; densitometry as in (C). For evidence against the role of the AP in leptin sensitization by CB₁ blockade, see Figure S5. Vertical bars represent SEM.

Figure 6. Peripheral CB₁R Inverse Agonism Reduces Leptin Expression and Secretion by Adipose Tissue

(A) JD5037 or SLV319 (3 mg/kg/day, 28 days) reverses the HFD-induced increase in adipose tissue leptin mRNA. *p < 0.05 relative to STD vehicle, ^#p < 0.05 relative to HFD-vehicle. n = 6 mice/group. (B) Activation of CB₁R in 3T3 L1 adipocytes increases leptin secretion. Cells were incubated for 24 hr with 100 nM noladin ether, 100 nM HU-210, or 500 nM anandamide in the presence of the FAAH inhibitor AM3506, 100 nM (Godlewski et al., 2010), alone or in combination with 100 nM JD5037. *p < 0.05 relative to agonist alone. (C) CB₁ agonists increase active Giα in 3T3 L1 adipocytes, as quantified by densitometry of western blots. (D) Pertussis toxin blocks CB₁ agonist-induced leptin secretion by 3T3 L1 adipocytes. (E and F) Peripheral CB₁ blockade reverses obesity-induced reduction of adipose tissue NE content and β3-receptor mRNA. *p < 0.05 relative to STD, ^#p < 0.05 relative to HFD vehicle. For the effect of JD5037 in denervated DIO mice, see Figure S6. Vertical bars represent SEM.

Figure 7. Reduced Leptin Clearance in DIO Mice Is Normalized by Peripheral CB₁ Blockade

(A) Pharmacokinetics of human leptin (10 mg/kg i.p.) injected into lean (STD-veh, t_1/2 33.5 ± 0.1 min) and DIO mice (HFD-veh, t_1/2 50.2 ± 4.8 min, p < 0.05) and DIO mice treated with JD5037, 3 mg/kg/day p.o. for 7 days (t_1/2 28.8 ± 0.9 min). Note that the increase in t_1/2 in DIO mice is reversed by JD5037, *p < 0.05 relative to STD, ^#p < 0.05 relative to HFD vehicle. (B) Leptin microuria in DIO mice is reversed by JD5037 treatment, * and ^# as in (A). (C) JD5037 reverses the diet-induced increase in serum creatinine levels, * and ^# as in (A). (D) Reduction in megalin mRNA in kidney of DIO mice is reversed by JD5037 treatment. (E) HFD reduces renal megalin protein in wild-type but not in CB₁^−/− mice (densitometry of western blots). *p < 0.05 relative to WT-STD, ^#p < 0.05 relative to CB₁^−/−-STD. (F) Reduced megalin and RAP protein in the DIO kidney are normalized by JD5037, 3 mg/kg/day for 28 days, as quantified by densitometry * and ^# as in (A). (G–I) Switching RPTEC from regular (RM) to serum-free medium (SF) increases megalin, RAP, and PPARα expression; reduces anandamide content; and increases leptin uptake. (J–L) In RPTEC maintained in regular medium, JD5037 (1 μM, 24 hr) increases megalin protein levels, leptin uptake, and leptin degradation, effects reversed by coincubation with 100 nM HU-210. *p < 0.05 relative to vehicle, ^#p < 0.05 relative to JD5037. For CB₁/megalin interaction in the choroid plexus, see Figure S7. Vertical bars represent SEM.