PMC

(A) Cumulative food intake of fasted Gucy2c^+/+ mice orally gavaged with 1 μg ST or the inactive alanine-substituted ST analogue, TJU, and refed HCD (n = 10 per group). (B) Cumulative food intake of fasted Gucy2c^+/+ mice injected i.v. with 1 μg ST or TJU and refed HCD (n = 10 per group). (C) Two-hour food intake of fasted Gucy2c^+/+ and Gucy2c^–/– mice injected i.v. with 1 μg TJU or ST and refed HCD (n = 10 per group). (D) Cumulative food intake of fasted Gucy2c^+/+ mice injected with TJU (1 μg) or ST and refed HCD (n = 10 per group). (E) Twelve-hour food intake of nonfasted Gucy2c^+/+ mice fed HCD and injected with 1 μg TJU or ST every 3 hours (n = 10 per group). (F) Two-hour food intake of fasted Gucy2c^+/+ and Gucy2c^–/– mice injected i.v. with TJU (1 μg) or PYY (3 μg) and refed HCD (n = 10 per group). (G) Two-hour food intake of fasted Gucy2c^+/+ mice injected i.v. with TJU (1 μg), ST (1 μg), or PYY (3 μg) and refed HCD (n = 10 per group). All data are mean ± SEM. **P < 0.01, ***P < 0.001.

(A) Growth curves of male Gucy2c^+/+ and Gucy2c^–/– mice raised on LCD (n = 20; P < 0.001). (B) Growth curves of female Gucy2c^+/+ and Gucy2c^–/– mice raised on HCD (n = 20–40; P < 0.01). (C) Adiposity index (percentage body fat) of 12-month-old mice (n = 10–13 per group). (D) Visceral adipose tissue (VAT), subcutaneous adipose tissue (SAT), and total adipose tissue of 12-month-old mice (n = 10–13 per group). (E) Quantification of lean body mass of 12-month-old mice raised on LCD or HCD (n = 10–13 per group). (F) Total body water content of mice determined by 72 hours desiccation at 90°C (n = 6). All data are mean ± SEM. ***P < 0.001.

(A) Liver triglyceride content of 12-month-old mice raised on LCD (n = 19). (B) H&E staining of representative left liver lobe sections of 12-month-old mice raised on LCD (scale bar: 200 μM). (C) Mean fasted serum leptin concentrations of mice raised on LCD (n = 6–10 per group). (D) Mean heart size (wet weight) of 12-month-old mice raised on LCD or HCD (n = 10–13 per group). (E) Mean fasted serum leptin concentrations of 12-month-old mice raised on HCD (n = 10). (F) Correlation of fasted serum leptin level and body weight of 12-month-old mice raised on HCD (r² = 0.47, P < 0.01). The diagonal line represents a linear regression model. (G) Mean fasted serum insulin concentrations of 12-month-old mice raised on HCD (n = 10). (H) Glucose tolerance test of fasted 12-month-old mice raised on HCD injected i.p. with glucose (2.5 mg/g) (n = 6). All data are mean ± SEM. NS = P > 0.05. *P < 0.05, **P < 0.01.

(A) Daily food consumption of 3-month-old female and male mice fed MCD or HCD. Points represent the mean of 10 daily food intake measurements (n = 10 per group). (B) Growth of female mice pair-fed HCD (2.3 g/mouse/d) (n = 12). (C) Correlation of mean daily food intake and weight gain of 4-month-old mice fed HCD during a 10-day period (r² = 0.72, P < 0.001) (n = 10 per group). The diagonal line represents a linear regression model. (D) Twenty-four–hour food intake of fasted 3- to 4-month-old female and male mice refed MCD or HCD (n = 10–20 per group). (E) Cumulative food intake of 3- to 4-month-old fasted mice refed HCD (n = 10 per group). (F) Two-hour food consumption of fasted mice gavaged with an isovolumetric bolus (300 μl) of water, olive oil, or 35% glucose (n = 5 per group). All data are mean ± SEM. Horizontal bars represent mean values. Scattergram points represent data for individual mice. *P < 0.05, **P < 0.01, ***P < 0.001.

(A) Free fatty acid content of feces of 3- to 4-month-old mice raised on HCD (n = 16). Scattergram points represent data for individual mice. (B and C) Serum triglyceride concentrations of fasted mice with or without tyloxapol (0.75 mg/g) after olive oil gavage (1.5 mg/g) (n = 5). (D) Levels of expression of metabolic genes in intestine, determined by qRT-PCR, normalized to Vil1 expression (n = 4–5 per group). (E) Daily activity patterns of Gucy2c^+/+ and Gucy2c^–/– mice (n = 6). (F) Core body temperatures of mice exposed to a 4°C environment for 24 hours (n = 6). All data are mean ± SEM. Horizontal bars represent mean values.

(A) Gucy2c expression in tissues of Gucy2c^+/+ mice, normalized to β-actin (Actb) expression (n = 4–6). The inset shows a representative gel image of the Gucy2c qRT-PCR products. (B) PCR products of the coding sequence of Gucy2c from cDNA prepared from Gucy2c^+/+ mouse tissues. Int, intestine; Hyp, hypothalamus; Lu, lung; Kid, kidney; Spl, spleen; NTC, nontemplate control. (C) Representative immunoblot of intestine and hypothalamic GUCY2C. (D) Intestine and hypothalamic GUCY2C protein content determined by immunoblot analyses (n = 3). (E) Guanylyl cyclase activity of membrane preparations with or without 1 μM ST (n = 4). (F) Two-hour food intake of fasted Gucy2c^+/+ mice after administration of TJU (10 μg), ST (10 μg), or exendin-4 (1 μg) into the third ventricle and refed HCD (n = 3 per group). (G) Hypothalamic expression of appetite-regulating neuropeptides, normalized to Actb expression, in fasted Gucy2c^+/+ mice injected i.v. with 1 μg TJU or ST every 2 hours for 8 hours (n = 8). All data are mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.

(A) Cumulative food intake of fasted Gucy2c^+/+ mice injected i.v. with TJU (10 μg), prouroguanylin (10 μg), or proguanylin (10 μg) and refed HCD (n = 10 per group). (B) cGMP production of CT26-GUCY2C cells treated with PBS, prouroguanylin (5 μg), proguanylin (5 μg), hypothalamic protein (350 μg), or combinations for 30 minutes (black bars, pH 5.5; red bars, pH 8.0; dark red bar, combined data of pH 5.5/8.0) (n = 4–10 per group). (C) Food intake and serum prouroguanylin concentrations of fasted Gucy2c^+/+ mice refed HCD (n = 7). (D) Serum prouroguanylin concentrations of human volunteers fasted for 12 hours and fed a standardized test meal (Supplemental Table 2) (P < 0.01, mean postprandial [15–150 minutes] level versus fasting [0 minutes] level) (n = 9). (E) Food intake of fasted Gucy2c^+/+ mice injected with PBS (control) or prouroguanylin antiserum (100 μl, 1:50 dilution) during the 1- to 2-hour interval after refeeding (n = 10 per group). All data are mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.