Catalytic antibody degradation of ghrelin increases whole-body metabolic rate and reduces refeeding in fasting mice

Abstract

Obesity is a chronic, costly, and globally prevalent condition, with excess caloric intake a suspected etiologic factor. Nonsurgical treatments are modestly efficacious, and weight loss maintenance is hampered by anti-famine homeostatic mechanisms. Ghrelin, a gastric hormone linked to meal initiation, energy expenditure, and fuel partitioning, is hypothesized to facilitate weight gain and impede weight loss. Unique among known animal peptides, the serine-3 residue of ghrelin is posttranslationally acylated with an n-octanoic acid, a modification important for the peptide's active blood-brain transport and growth hormone secretagogue receptor-1 agonist activity. Pharmacological degradation of ghrelin would be hypothesized to reduce ghrelin's biological effects. To study endogenous ghrelin's role in appetite and energy expenditure, we generated antibodies that hydrolyze the octanoyl moiety of ghrelin to form des-acyl ghrelin. The most proficient antibody catalyst, GHR-11E11, was found to display a second-order rate constant of 18 M(-1) x s(-1) for the hydrolysis of ghrelin to des-acyl ghrelin. I.v. administration of GHR-11E11 (50 mg/kg) maintained a greater metabolic rate in fasting C57BL/6J mice as compared with mice receiving a control antibody and suppressed 6-h refeeding after 24 h of food deprivation. Indirect respiratory measures of metabolism after refeeding and relative fuel substrate utilization were unaffected. The results support the hypothesis that acylated ghrelin stimulates appetite and curbs energy expenditure during deficient energy intake, whereas des-acyl ghrelin does not potently share these functions. Catalytic anti-ghrelin antibodies might thereby adjunctively aid consolidation of caloric restriction-induced weight loss and might also be therapeutically relevant to Prader-Willi syndrome, characterized after infancy by hyperghrelinemia, hyperphagia, and obesity.

Fig. 1.

Structures of ghrelin-derived peptides 1–10 synthesized for the generation, specificity and kinetic characterization of catalytic mAbs.

Fig. 2.

Kinetic evaluation of catalyzed ghrelin hydrolysis. (Left) Catalysis of ghrelin hydrolysis by addition of 1 μM GHR-11E11 to varying concentrations of substrate 3 (0.4–50 μM) at 37 °C. (Right) Dose-response plot of inhibiton of ghrelin hydrolysis by 1 μM GHR-11E11 with varying inhibitor 1 concentrations; IC₅₀: 21.3μM.

Fig. 3.

Shown are the rate of energy expenditure (heat, Top Left), respiratory exchange ratio (RER) (Top Right), the rates of oxygen consumption (VO₂) (Middle Left) and carbon dioxide production (VCO₂) (Middle Right), and rates of horizontal and vertical motor activity (Bottom) in food-deprived, antibody-treated, adult male C57BL/6J mice tested in open-circuit indirect calorimetry chambers. Data are expressed in 2 h bins as M ± SEM across the 12-h light cycle. Mice received i.v. administration (i.v. 50 mg/kg) of a catalytic antibody against ghrelin (n = 8, GHR-11E11) or of an isotype-matched nicotine control Ab (n = 9, NIC-1 9D9) before data collection; *, P < 0.05 vs. control Ab-treated mice.

Fig. 4.

Food intake in 24-h food-deprived adult male C57BL/6J mice that had received i.v. administration (i.v. 50 mg/kg) of a catalytic antibody against ghrelin (n = 8, GHR-11E11) or of an isotype-matched nicotine control Ab (n = 9, NIC-1 9D9) 24 h earlier. Data express M ± SEM cumulative food intake across 6 h of refeeding beginning from the light cycle onset. *, P < 0.05 vs. control Ab-treated mice.

Fig. 5.

Panels show the rate of energy expenditure (heat) (Top Left), respiratory exchange ratio (RER) (Top Right), rates of oxygen consumption (VO₂) (Bottom Left), and carbon dioxide production (VCO₂) (Bottom Right) in adult male C57BL/6J mice residing in open-circuit indirect calorimetry chambers. Data were collected in a 24-h food-deprived state (“Unfed”) and during 6 h of refeeding on chow. Data are expressed in 1 h bins as M ± SEM. Mice had received i.v. administration (i.v. 50 mg/kg) of a catalytic antibody against ghrelin (n = 8, GHR-11E11) or of an isotype-matched nicotine control Ab (n = 9, NIC-1 9D9) 24 h before data collection; *, P < 0.05 vs. control Ab-treated mice.