Cholecystokinin responsiveness varies across the population dependent on metabolic phenotype

Abstract

Background: Cholecystokinin (CCK) is an important satiety factor, acting at type 1 receptors (CCK1Rs) on vagal afferent neurons; however, CCK agonists have failed clinical trials for obesity. We postulated that CCK1R function might be defective in such patients due to abnormal membrane composition, such as that observed in cholesterol gallstone disease.Objective: Due to the challenges in directly studying CCK1Rs relevant to appetite control, our goal was to develop and apply a method to determine the impact of a patient's own cellular environment on CCK stimulus-activity coupling and to determine whether CCK sensitivity correlated with the metabolic phenotype of a high-risk population.Design: Wild-type CCK1Rs were expressed on leukocytes from 112 Hispanic patients by using adenoviral transduction and 24-h culture, with quantitation of cholesterol composition and intracellular calcium responses to CCK. Results were correlated with clinical, biochemical, and morphometric characteristics.Results: Broad ranges of cellular cholesterol and CCK responsiveness were observed, with elevated cholesterol correlated with reduced CCK sensitivity. This was prominent with increasing degrees of obesity and the presence of diabetes, particularly when poorly controlled. No single standard clinical metric correlated directly with CCK responsiveness. Reduced CCK sensitivity best correlated with elevated serum triglycerides in normal-weight participants and with low HDL concentrations and elevated glycated hemoglobin in obese and diabetic patients.Conclusions: CCK responsiveness varies widely across the population, with reduced signaling in patients with obesity and diabetes. This could explain the failure of CCK agonists in previous clinical trials and supports the rationale to develop corrective modulators to reverse this defective servomechanism for appetite control. This trial was registered at www.clinicaltrials.gov as NCT03121755.

Keywords: cholecystokinin; cholesterol; epidemiology; hormonal responsiveness; obesity drug development; population study.

FIGURE 1

Distribution of CCK sensitivity in the patient population and its correlation with cellular cholesterol. Shown is a typical CCK concentration-response curve generated by measuring increase in intracellular calcium concentrations in leukocytes 24 h after transduction (A). The inset in panel A shows a representative image of the efficiency CCK1R expression achieved within this time as measured by GFP fluorescence. Histograms of the CCK EC₅₀ (B) and cellular cholesterol (C) distributions, with density curves for the total population included in the study (n = 112). Correlation plots between the CCK EC₅₀ on the x axis and cellular cholesterol (r = 0.4051, P = 0.000009) (D) or CCK1R expression level as measured by GFP fluorescence (r = −0.005468, P = 0.9569) (E) on the y axis. Spearman’s correlation test was used to obtain the r and P values. CCK, cholecystokinin; CCK1R, type 1 cholecystokinin receptor; Chol, cholesterol; EC₅₀, concentration stimulating one-half of the maximal response; GFP, green fluorescent protein; max, maximum; RFU, relative fluorescence unit.

FIGURE 2

Correlations between CCK sensitivity and cellular cholesterol in different patient populations. Shown are the comparisons for the correlation between the potency of CCK responses (EC₅₀) and cellular cholesterol along with the respective n values, Spearman correlation coefficients (r), and P values for all patients (A) and patients selected on the basis of sex (B and C), obesity (D–F), components of metabolic syndrome (in patients without diabetes) (G–K), metabolic syndrome (in patients without diabetes) (L), and the absence or presence of diabetes (M and N). Values significant at P < 0.05 include correlations in all patients in the cohort, women, and patients who were overweight or obese and morbidly obese, with a large waist size, with elevated triglycerides, with elevated FG, with reduced HDL cholesterol, and both nondiabetic and diabetic patients. There were 21 patients in the metabolic syndrome group who were nondiabetic and fulfilled the American Heart Association criteria for metabolic syndrome, including ≥3 of the following 5 criteria: waist circumference ≥101.6 cm in men and ≥88.9 cm in women, triglyceride concentration of ≥150 mg/dL, HDL concentrations ≤40 mg/dL in men and ≤50 mg/dL in women, FG ≥100 mg/dL, and BP ≥130/85 mm Hg. Spearman’s correlation test was used to obtain the r and P values. BP, blood pressure; CCK, cholecystokinin; EC₅₀, concentration stimulating one-half of the maximal response; FG, fasting glucose; TG, triglycerides.

FIGURE 3

Multivariate models to predict CCK EC₅₀ values in patient populations. (A) Actual and predicted CCK sensitivity (EC₅₀ values) among diabetic participants based on a model that included HDL cholesterol and glycated hemoglobin as predictor variables (line represents the unity line where the actual CCK EC₅₀ equals the predicted CCK EC₅₀; n = 35; r² = 0.26, P < 0.01). (B) Actual CCK sensitivity data by leukocyte cellular cholesterol values with plotting symbols for men (n = 43) and women (n = 69) in the entire population (lines represent predicted CCK EC₅₀ based on a model that included sex, leukocyte cellular cholesterol, and sex-by-leukocyte cellular cholesterol interaction). The overall r² value for the model was 0.29 (P < 0.001). Sex subgroupings without a cell cholesterol-by-sex interaction term included estimates of r² = 0.40 (P < 0.001) for women and r² = 0.06 (P = 0.12) for men. CCK, cholecystokinin; EC₅₀, concentration stimulating one-half of the maximal response.