Oligoclonal antibody targeting ghrelin increases energy expenditure and reduces food intake in fasted mice

Abstract

Ghrelin, an enteric peptide hormone linked to the pathophysiology of obesity has been a therapeutic target of great interest over the past decade. Many research efforts have focused on the antagonism of ghrelin's endogenous receptor GHSR1a, which is found along ascending vagal afferent fibers, as well as in the arcuate nucleus of the hypothalamus. Additionally, peptidic inhibitors of ghrelin O-acyltransferase, the enzyme responsible for the paracrine activation of ghrelin, have recently been studied. Our research has taken an alternative immunological approach, studying both active and passive vaccination as a means to sequester ghrelin in the periphery, with the original discovery in rat of decreased feed efficiency and adiposity, as well as increased metabolic activity. Using our previous hapten designs as a stepping-stone, three monoclonal antibodies (JG2, JG3, and JG4) were procured against ghrelin and tested in vivo. While mAb JG4 had the highest affinity for ghrelin, it failed to attenuate the orexigenic effects of food deprivation on energy metabolism or food intake in mice. However, animals that were administered a combination of JG3:JG4 (termed a doublet) or JG2:JG3:JG4 (termed a triplet) demonstrated higher heat dispersion and rate of respiration (higher CO(2) emission and O(2) consumption) during a 24 h fast refeed. Mice administered the triplet cocktail of JG2:JG3:JG4 also demonstrated decreased food intake upon refeeding as compared to control animals. Recently, Lu and colleagues reported that a passive approach using a single, high affinity N-terminally directed monoclonal antibody did not abrogate the effects of endogenous ghrelin. Our current report corroborates this finding, yet, refutes that a monoclonal antibody approach cannot be efficacious. Rather, we find that a multiple monoclonal antibody (oligoclonal) approach can reproduce the underlying logic to previously reported efficacies using active vaccinations.

Fig. 1

Shown is the secondary structure of full-length acyl-ghrelin with potential binding sites of mABs JG2, JG3, and JG4, inferred from BIAcore analysis, highlighted as oval regions. mAB JG4 recognizes the N-terminus of ghrelin, specifically the acylated side chain found on serine-3. mAB JG3 recognizes an internal binding site on ghrelin, possibly at the loop structure found from residues serine-18 – lysine-20, and mAB JG2 recognizes the C-terminus of ghrelin. Structure shown modified from PDB 1P7X.

Fig. 2

Shown are the rate of energy expenditure (heat, Top Left), respiratory exchange ratio (RER, Top Right), the rates of carbon dioxide production (VCO₂, Bottom Left) and oxygen consumption (VO₂, Bottom Right) in food deprived, antibody-treated, adult male C57BL/6J mice as recorded in open-circuit indirect calorimetry chambers. Data are expressed in 2-hr bins as M ± SEM during the first 16 hrs of fasting beginning at light onset. Mice received subcutaneous administration (15 mg/kg total dose) of ghrelin mAbs in doublets ● JG2:JG4, JG3:JG4, and JG2:JG3 (n=6) or a nicotine control Ab (n=6, NIC-1 9D9) 4 days prior to data collection; *, p < 0.05 vs. control Ab-treated mice. Dark onset begins at hrs 13–14 of the fasting stage.

Fig. 3

Shown are the rate of energy expenditure (heat, Top Left), respiratory exchange ratio (RER, Top Right), the rates of carbon dioxide production (VCO₂, Bottom Left) and oxygen consumption (VO₂, Bottom Right) in antibody-treated, adult male C57BL/6J mice refeeding after a 24-hr fast as recorded in open-circuit indirect calorimetry chambers. Data are expressed in 1-hr bins as M ± SEM during the first 6 hrs of refeeding beginning at light onset. Mice received subcutaneous administration (15 mg/kg total dose) of ghrelin mAbs in doublets ● JG2:JG4, JG3:JG4, and JG2:JG3 (n=6) or a nicotine control Ab (n=6, NIC-1 9D9) 5 days prior to data collection; *, p < 0.05, **, p < 0.01 vs. control Ab-treated mice.

Fig. 4

Food intake in 24-hr food-deprived, antibody-treated adult male C57BL/6J mice as recorded in open-circuit indirect calorimetry chambers. Data are expressed in 30 min bins as M ± SEM during the first 6 hrs of refeeding beginning at light onset. Mice received subcutaneous administration (15 mg/kg total dose) of ghrelin mAbs in doublets ● JG2:JG4, JG3:JG4, and JG2:JG3 (n=6) or a nicotine control Ab (n=6, NIC-1 9D9) 5 days prior to data collection.

Fig. 5

Shown are the rate of energy expenditure (heat, Top Left), respiratory exchange ratio (RER, Top Right), the rates of carbon dioxide production (VCO₂, Bottom Left) and oxygen consumption (VO₂, Bottom Right) in antibody-treated, adult male C57BL/6J mice as recorded in open-circuit indirect calorimetry chambers. Data are expressed in 1-hr bins as M ± SEM during the last hr of the fasting stage (“Unfed”) and the first 6 hrs of refeeding beginning at light onset. Mice received subcutaneous administration (15mg/kg total dose) of ghrelin mAbs in triplet ● ghr mAbs JG2, JG3, & JG4 (n=6) or a -■- nicotine control Ab (n=6, NIC-1 9D9) 5 days prior to data collection; *, p < 0.05 vs. control Ab-treated mice.

Fig. 6

Food intake in 24-hr food-deprived, antibody-treated adult male C57BL/6J mice as recorded in open-circuit indirect calorimetry chambers. Data are expressed in 30 min bins as M ± SEM during the first 6 hrs of refeeding beginning at light onset. Mice received subcutaneous administration (15 mg/kg total dose) of ghrelin mAbs in triplet ● ghr mAbs JG2, JG3, & JG4 (n=6) or a -■- nicotine control Ab (n=6, NIC-1 9D9) 5 days prior to data collection; ***, p < 0.0001 vs. control Ab-treated mice.