Fig 1.
The effect of FOY-251 on gallbladder weights in genetically CCK1R deficient mice or CCK1R blockade with loxiglumide.
(A) Gallbladder weights 15 minutes after oral vehicle (cyan bars) or FOY-251 (green bars) administration to fasted WT control (closed circles) and CCK1R KO (open circles) mice. (B) Effect of increasing doses of oral loxiglumide (beige bars) on gallbladder weight in fasted C57BL/6 mice. (C) Effect of FOY-251 (green bar), loxiglumide (beige bar), or both (green and beige cross-hatched bar) on gallbladder weight in fasted C57BL/6 mice. Animals were first treated with oral vehicle or loxiglumide (t = 0 min), followed by oral vehicle or FOY-251 (t = 30 min); gallbladder weight was measured at t = 45 min. Gallbladders from four fasted naïve mice (grey bar) were included to demonstrate an absence of effect of vehicle treatment. Values are expressed as mean ± SEM; *p < 0.05 for the indicated comparisons.
Fig 2.
Effect of FOY-251 and/or loxiglumide on gastric emptying and food intake in fasted DIO mice.
(A) Blood acetaminophen appearance following treatment with 100 mg/kg oral FOY-251, 30 mg/kg loxiglumide, or their combination (N = 7-8/group). 20 μg SC exendin-4 was used as a positive control to validate the experimental model (N = 5). All animals received oral gavage or subcutaneous injection at the following timepoints: oral vehicle (20%HP-β-CD) or loxiglumide (t = −40 min), oral vehicle (5%HPMC) or FOY-251 (t = −30 min), subcutaneous PBS or exendin-4 (t = −20 min), followed by oral acetaminophen (100 mg/kg) at t = 0 min. *p < 0.05 vs vehicle control group. (B) Integrated blood acetaminophen for data shown in panel (A). (C) 4-hour food intake in DIO mice administered oral vehicle or loxiglumide (t = −60 min), followed by oral vehicle, FOY-251 or Rimonabant at t = −30 min. Food consumption during the dark phase was measured over 4-hours starting at t = 0 min (N = 8/group). Values are expressed as mean ± SEM; *p < 0.05 for the indicated comparisons.
Fig 3.
Chronic food intake and body weight effects of FOY-251 administered as high fat diet (HFD) admixture to WT and CCK1R KO.
(A) Daily FOY-251 compound dose calculated using diet consumption and body weight. FOY-251 was formulated at 1.59 mg/gm of 60% HFD. (B) Daily food intake WT and CCK1R KO mice provided with non-medicated diet (Control), HFD with FOY-251, and animals pair-fed (PF) non-medicated diet at the same amount as that consumed by respective FOY-251 treated groups. ^p < 0.05 WT-Control vs WT-FOY-251; $p < 0.05 WT-Control vs WT-PF; *p < 0.05 CCK1R KO-Control vs CCK1R KO-FOY-251; #p < 0.05 CCK1R KO-Control vs CCK1R KO-PF. Weekly cumulative food intake over 4 weeks in wild type mice (C) or CCK1R KO mice (D) treated with FOY-251 (green bars), or non-medicated HFD Control (cyan bars) or animals pair fed to the same level as FOY-251 treated mice (black bars). *p < 0.05 Control vs FOY-251 for each genotype. Percent change in body weight in WT (E) and CCK1R KO (F) mice treated with FOY-251, non-medicated HFD Control or pair-fed (PF) non-medicated HFD at the same level as FOY-251-treated group. *p < 0.05 vs Control. Data represent Mean±SEM of 9-10 animals per group.
Fig 4.
Effect of chronic FOY-251 treatment on body composition and fecal protein excretion in WT and CCK1R KO diet-induced obese mice.
Body composition of WT and CCK1R KO mice fed control HFD, FOY-251 HFD admixture, and animals pair-fed to the FOY-251 group was measured by whole body MRI to measure fat mass (A) and lean mass (B). Data represent change in body composition from baseline for each individual animal. Cumulative protein in excreted feces over the 4 weeks of treatment was normalized to total fecal output (C) or presented as absolute total protein excreted (D) in WT and CCK1R KO mice receiving non-medicated high fat diet, FOY-251 HFD admixture or pair fed non-medicated HFD. (E) Weekly cumulative total fecal protein excretion over the 4-week study period in CCK1R KO mice and WT controls. Data represent the mean±SEM of 8-10 animals per group; *p < 0.05 for the indicated comparisons.
Fig 5.
Liver steatosis in chronic FOY-251-treated DIO CCK1R KO mice and their wild type controls.
(A) Post-mortem organ fat content measured using MRI in WT and KO mice fed control HFD, FOY-251 HFD admixture, and animals pair-fed to the FOY-251 group. (B) Representative hematoxylin & eosin stained liver tissue sections from WT and CCK1R KO mice; blue sections indicate the software analysis algorithm determined tissue area for representative CCK1R KO samples. Black scale bars represent 500μm. (C) Quantitative image analysis for steatosis measuring vacuolar burden (%vacuole area/liver area) using Visiopharm software. (D) Semiquantitative blinded histopathology grading of liver vacuolation (distinct clear vacuoles of variable size, with displacement of nucleus) consistent with lipidosis (steatosis) severity: [minimal (grade 1), mild (grade 2), moderate (grade 3), marked (grade 4) and severe (grade 5)]. *p < 0.05 for indicated comparisons.
Fig 6.
Glucose homeostasis in chronic FOY-251-treated DIO CCK1R KO mice and their wild type controls.
Fasted blood glucose (A) and plasma insulin levels (B) were obtained on day 27 of treatment following a 5-hour fast in WT mice (filled symbols) and CCK1R KO mice (open symbols) given non-medicated high fat diet (circles), HFD formulated with FOY-251 (squares), or animals pair-fed non-medicated diet (triangles) at the same amount consumed by FOY-251 treated mice. *p < 0.05 for indicated comparisons. (C) Plasma FGF-21 was measured in terminal fed plasma samples taken during sacrifice after 4-weeks of treatment. Data represent mean±SEM of 9-10 animals per group.
Fig 7.
Role of CCK in mediating metabolic effects of Enteropeptidase/Trypsin inhibition with Camostat Metabolite FOY-251.
Inhibition of EP/T causes weight loss by caloric excretion via protein in feces, and also partially via CCK release from the small intestine. CCK acts centrally to reduce food intake, as well as on CCK1R receptors to reduce gastric emptying and increase bile acid secretion. Results from the current study indicated CCK1R-dependent and -independent effects of EP/T inhibition on weight loss in mice, with concomitant improvement of steatosis.