EP3R–/– Mice Showed Increased Body Weight Caused by Increased Fat Storage. Male EP3R–/– mice fed ad libitum on an 11% fat diet exhibited an increase in body weight that resulted in an obese phenotype (Fig. 1A). EP3R–/– body weights showed a gradual increase, with continuous accumulation up to 20% above that of WT littermate controls by week 20 (t = 4.41; P = 0.001) and continuing thereafter up to a recorded 30% increase by week 40 (t = 4.7; P = 0.006) (Fig. 1B). Body weight gain was primarily attributable to increased adipose tissue deposition (Fig. 1C). Comparison of the body composition of 3- and 6-month-old mice revealed that the early deposition of intraabdominal fat pads (gonadal, retroperitoneal, and mesenteric) in EP3R–/– mice at 
3 months was followed by significant deposition of fat in adjacent tissues that include the liver and subcutaneous fat pads (inguinal and the groin) in EP3R–/– mice at 6 months (Fig. 1D). At 3 and 6 months of age, abdominal fat accounted for 6.62 ± 0.47% and 8.59 ± 0.25% of the total body weight in EP3R–/– mice compared with the 3.37 ± 0.69% and 3.46 ± 0.16% in the WT mice, respectively (Fig. 1C). Subcutaneous fat pads at both ages accounted for 2.49 ± 0.33% and 5.2 ± 0.28% of the total body weight in EP3R–/– mice compared with the 1.99 ± 0.32% and 1.47 ± 0.12% in the WT mice, respectively (Fig. 1D). No significant differences in the brown adipose tissue percentage of body weight were seen between EP3R–/– and WT mice.
EP3R–/– Mice Showed High Serum Levels of Insulin and Leptin at 3 and 6 Months of Age. Measurements of circulating levels of insulin and leptin demonstrated that EP3R–/– mice had significantly elevated levels of both hormones. At 3 and 6 months of age, insulin levels accounted for 12.30 ± 1.37 ng/ml and 14.50 ± 1.37 ng/ml in EP3R–/– mice compared with 6.95 ± 1.44 ng/ml and 11.01 ± 1.96 ng/ml in the WT mice during the light cycle (insulin, t = 2.3, P = 0.042, EP3R–/– mice vs. WT mice at 3 months of age; insulin, t = 1.4, P = 0.187, EP3R–/– mice vs. WT mice at 6 months of age); insulin levels accounted for 15.43 ± 1.5 ng/ml and 16.49 ± 0.65 ng/ml in EP3R–/– mice compared with 11.01 ± 1.96 ng/ml and 6.18 ± 3.91 ng/ml in the WT mice during the dark cycle (insulin, t = 2.74, P = 0.033, EP3R–/– mice vs. WT mice at 3 months of age; insulin, t = 4.7, P = 0.003, EP3R–/– mice vs. WT mice at 6 months of age) (Fig. 2Upper).
Leptin levels were higher in EP3R–/– mice compared with WT mice during both the light and dark cycles. At 3 and 6 months of age, leptin levels were 19.61 ± 2.79 ng/ml and 22.94 ± 2.43 ng/ml in EP3R–/– mice compared with 10.18 ± 1.45 ng/ml and 7.17 ± 1.03 ng/ml in the WT mice during the light cycle (leptin, t = 3.0, P = 0.013, EP3R–/– mice vs. WT mice at 3 months of age; leptin, t = 4.8, P = 0.0019, EP3R–/– mice vs. WT mice at 6 months of age); leptin levels were 21.27 ± 0.95 ng/ml and 25.07 ± 1.47 ng/ml in EP3R–/– mice compared with 7.16 ± 1.03 ng/ml and 2.78 ± 0.11 ng/ml in the WT mice during the dark cycle (leptin, t = 9.1, P = 0.0001, EP3R–/– mice vs. WT mice at 3 months of age; leptin, t = 10.26, P = 0.0001, EP3R–/– mice vs. WT mice at 6 months of age) (Fig. 2 Lower).
EP3R–/– Mice Showed Impaired Glucose Tolerance and Insulin Resistance. At 3 months of age, the increased adiposity in EP3R–/– mice was accompanied by abnormalities in glucose metabolism. Even though mice were fasted for 24 h before the glucose-tolerance test, basal levels of glucose in EP3R–/– mice were significantly higher compared with WT mice (EP3R–/–, 149.25 ± 15.91 mg/dl; WT, 103.01 ± 10.1 mg/dl; t = 2.45; P = 0.04) (Fig. 3A). After the mice were challenged with a 1.5 mg of glucose per gram of body weight, serum glucose concentrations were similarly elevated at 15 min in EP3R–/– and WT mice, but serum glucose concentrations reached higher levels in EP3R–/– mice at 30 min (445 ± 21.08 mg/dl) than in the WT mice (381 ± 22.54 mg/dl) (t = 2.08; P = 0.04). At 60 min after the glucose challenge, glucose levels were similar in both EP3R–/– and WT mice, but higher levels in EP3R–/– mice were reached again at 120 min (EP3R–/–, 242.25 ± 19.55 mg/dl; WT, 136.25 ± 20.42 mg/dl; t = 3.78; P = 0.0091) and 180 min (EP3R–/–, 157.5 ± 6.38 mg/dl; WT, 119.24 ± 3.72 mg/dl; t = 5.17; P = 0.0021) (Fig. 3A).
At 6 months of age, abnormalities in glucose metabolism were significantly higher in EP3R–/– mice than at 3 months of age (Fig. 3B). Basal levels of glucose in EP3R–/– mice were significantly higher compared with WT mice (EP3R–/–, 196.4 ± 22.84 mg/dl; WT, 105.82 ± 6.17 mg/dl; t = 4.16; P = 0.0024). After a glucose challenge, EP3R–/– mice showed higher glucose levels at 15 min (465.2 ± 22.05 mg/dl) than did WT mice (382.5 ± 22.31 mg/dl) (t = 2.6; P = 0.0283). The same was true at 30 min (EP3R–/–, 487.8 ± 11.7 mg/dl; WT, 404.83 ± 33.35 mg/dl; t = 2.26; P = 0.049), 60 min (EP3R–/–, 420.6 ± 23.54 mg/dl; WT, 282 ± 22.15 mg/dl; t = 4.27; P = 0.0021), 120 min (EP3R–/–, 286.1 ± 25.93 mg/dl; WT, 233.65 ± 16.75 mg/dl; t = 1.7; P = 0.1135), and 180 min (EP3R–/–, 184.6 ± 15.14 mg/dl; WT, 146.5 ± 9.21 mg/dl; t = 2.24; P = 0.048) (Fig. 3B).
We determined the ability of insulin to acutely stimulate glucose disposal or clearance by performing an acute insulin challenge in EP3R–/– and WT mice at both 3 and 6 months of age. At 3 months of age, the ability of insulin to acutely stimulate glucose disposal in EP3R–/– mice was significantly blunted at 15 min, indicating a short-term decrease in insulin sensitivity (EP3R–/–, 158.3 ± 10.59 mg/dl; WT, 125.8 ± 11.79 mg/dl, which corresponded to a 84.36% and 63.63% reduction in baseline values of EP3R–/– and WT, respectively; P = 0.0246) (Fig. 3 C and C’). At 6 months of age, basal levels of glucose were higher in EP3R–/– mice (EP3R–/–, 287.25 ± 48.32 mg/dl; WT, 173.02 ± 17.16 mg/dl), and acute insulin challenge was unable to stimulate glucose disposal in EP3R–/– mice at 15 min (EP3R–/–, 265 ± 53.71 mg/dl; WT, 125.3 ± 16.04 mg/dl, which corresponded to a 90.45% and 73.01% reduction in baseline values of EP3R–/– and WT, respectively; P = 0.046). Even though values at 30 min (EP3R–/–, 280.12 ± 48.5 mg/dl; WT, 148.02 ± 13.21 mg/dl), 60 min (EP3R–/–, 274.07 ± 41.69 mg/dl; WT, 156.23 ± 22.43 mg/dl), or 120 min (EP3R–/–, 290.94 ± 44.14 mg/dl; WT, 185.16 ± 28.89 mg/dl) were significantly higher in EP3R–/– mice, comparisons of the percentage of change from their own baseline were not significant (Fig. 3 D and D’).
EP3R–/– Mice Exhibited Increased Motor Activity and Core Body Temperature. Radiotelemetric evaluation of motor activity indicates that EP3R–/– mice displayed peaks of increased motor activity during the light part of the day, when mice usually spend most of their time sleeping. Increased nocturnal activity was recorded continuously over 21 days in six EP3R–/– mice and six WT littermates. Continuous motor activity profile during 21 days is shown (Fig. 4A), and this profile demonstrates that the peaks of nocturnal activity in EP3–/– mice are not episodic but instead are recurrent daily events occurring with a frequency of between two and four episodes per night (Fig. 4 A and C). The increase in motor activity during the light cycle in EP3R–/– mice compared with WT mice was punctuated and separated by phases in which the motor activity of the EP3R–/– mice was indistinguishable from that of the WT mice (Fig. 4 B and C). However, cumulative analysis demonstrated that these peaks contributed to an overall 60.3% increased motor activity in EP3R–/– mice. Increased motor activity in EP3R–/– mice was also observed during the dark cycle, and this increase accounted for 24.73% of the motor activity (Fig. 4B). Core body temperature of EP3R–/– mice was slightly elevated during the corresponding peaks of increased motor activity (see Fig. 4D).
EP3R–/– Mice Showed Increased Food Consumption. Measurement of food consumption in EP3R–/– and WT mice demonstrated that EP3R–/– ate significantly more food than the WT littermates at both ages. At 3 months of age, hourly food intake was similar between EP3R–/– and WT mice during the light cycle. However, EP3R–/– mice during the dark period showed an increase in food intake characterized by a continuous feeding (monophasic), whereas WT littermate controls showed an increase in feeding followed by a period of temporary decrease and then by a second increase in feeding, making the pattern biphasic (Fig. 5A). As a consequence, statistical differences are only observed in the dark period at 0:00 and 1:00 a.m. (0:00: EP3R–/–, 0.338 ± 0.02 g; WT, 0.236 ± 0.03 g; t = 2.35; P = 0.0466; 1:00: EP3R–/–, 0.298 ± 0.03 g; WT, 0.184 ± 0.03 g; t = 2.33; P = 0.0481). At 6 months of age, the increase in feeding strongly correlated with the peaks of activity and elevated temperature during both the light/inactive and the dark/active part of the day (Fig. 5B). Cumulative food intake was higher in EP3R–/– mice in both periods. During the light phase, significant differences were at 7:00 a.m. (EP3R–/–, 0.19 ± 0.03 g; WT, 0.09 ± 0.02 g; t = 2.25; P = 0.0379), 9:00 a.m. (EP3R–/–, 0.17 ± 0.04 g; WT, 0.05 ± 0.01 g; t = 2.51; P = 0.022), 15:00 p.m. (EP3R–/–, 0.16 ± 0.02 g; WT, 0.04 ± 0.02 g; t = 3.27; P = 0.004), and 17:00 p.m. (EP3R–/–, 0.17 ± 0.06 g; WT, 0.03 ± 0.01 g; t = 2.14; P = 0.047). During the dark phase, significant differences were at 0:00 a.m. (EP3R–/–, 0.36 ± 0.03 g; WT, 0.17 ± 0.04 g; t = 3.08; P = 0.006) and 1:00 a.m. (EP3R–/–, 0.27 ± 0.03 g; WT, 0.15 ± 0.03 g; t = 2.2; P = 0.041).
Both EP3R–/– and WT mice underwent food deprivation for 24 h, and subsequent food intake was measured every 12 h. Baseline measurement for 3 days confirmed differences in food intake. Food deprivation increased food intake in the next dark and light period in both strains, and full recovery was reached on day 4 (Fig. 5B).
