D by a 3V injection of NPY (0.2 mg/kg BW) or artificial cerebrospinal fluid (control). Plasma triglyceride (TG) levels were determined at indicated time points (A). VLDL-TG production rate was calculated from the slopes of the individual TG-time graphs (B). At t = 120 min, mice were exsanguinated and VLDL fractions were isolated from serum by ultracentrifugation. 35S-apoB production was determined by scintillation counting of the isolated VLDL fraction (C). Values are means 6 SD (n = 9212). doi:10.1371/journal.pone.0055217.gIn conclusion, acute central administration of NPY increases food intake without affecting hepatic VLDL production in mice, whereas NPY increases both food intake and VLDL production in rats. This apparent species difference in the effects of NPY, specifically on hepatic VLDL-TG production, is of great significance for future animal studies on the central regulation of hepatic VLDL production and underscores a general concern in animal research in view of extrapolating findings from specific animal studies to explain observations done in humans.Materials and Methods AnimalsFor all experiments, naive 15 weeks old male C57Bl/6J mice were used, housed in a temperature and humidity-controlled environment with free access to food and water. Experiments wereCentral NPY and Hepatic VLDL Production in Miceperformed after 4 h of fasting at 12:00 pm with food withdrawn at 8:00 am, unless indicated otherwise. Food intake and body weight were measured weekly during experiments. All animal experiments were approved by the Animal Ethics Committee of the Leiden University Medical Center, Leiden, The Netherlands.Intracerebroventricular SurgeryFor i.c.v. cannula implantation, mice were anaesthetized with 0.5 mg/kg BW Medetomidine (Pfizer, Capelle a/d IJssel, The Netherlands), 5 mg/kg BW Midazolam (Roche, Mijdrecht, The Netherlands) and 0.05 mg/kg BW Fentanyl (Janssen-Cilag, Tilburg, The Netherlands) and placed in a stereotactic device (TSE systems, Homburg, Germany). A 25-gauge guide cannula was implanted into the left lateral ventricle using the following coordinates from Bregma: 1.0 mm lateral, 0.46 mm posterior and 2.2 mm ventral. For third ventricle cannulations the following coordinates from Bregma were used: 0.0 mm lateral, 1.3 mm posterior and 5.7 mm ventral. The guide cannula was secured to the skull surface with dental 58-49-1 chemical information cement (GC Europe N.V., Leuven, Belgium) and the anesthesia was antagonized using 2.5 mg/kg BW Antipamezol (Pfizer, Capelle a/d IJssel, The Netherlands), 0.5 mg/kg BW Flumazenil (Roche, Mijdrecht, The Netherlands) and 1.2 mg/kg BW Naloxon (Orpha, 1662274 Purkersdorf, Austria). Animals were single housed after the surgery.vehicle (PBS, 100 mL). Both drugs were tested once, in the number of mice indicated. Blood samples were taken from the tail tip into chilled heparin-coated capillaries (Vitrex Medical, Herlev, Denmark) at the indicated time points up to 90 min after tyloxapol injection. The tubes were kept on ice after which they were centrifuged (12.000 rpm for 5 min at 4uC). Plasma TG concentration was determined using a commercially available kit according to the instructions of the manufacturer (no. 11488872, Roche Molecular MedChemExpress Fexinidazole Biochemicals, Indianapolis, IN) At 120 min, the animals were sacrificed and blood was collected by orbital puncture for isolation of VLDL by density gradient ultracentrifugation [36]. 35S-activity was measured in the VLDL fraction and VLDL-apoB production rate was calculated as dpm.h21 [37].Ve.D by a 3V injection of NPY (0.2 mg/kg BW) or artificial cerebrospinal fluid (control). Plasma triglyceride (TG) levels were determined at indicated time points (A). VLDL-TG production rate was calculated from the slopes of the individual TG-time graphs (B). At t = 120 min, mice were exsanguinated and VLDL fractions were isolated from serum by ultracentrifugation. 35S-apoB production was determined by scintillation counting of the isolated VLDL fraction (C). Values are means 6 SD (n = 9212). doi:10.1371/journal.pone.0055217.gIn conclusion, acute central administration of NPY increases food intake without affecting hepatic VLDL production in mice, whereas NPY increases both food intake and VLDL production in rats. This apparent species difference in the effects of NPY, specifically on hepatic VLDL-TG production, is of great significance for future animal studies on the central regulation of hepatic VLDL production and underscores a general concern in animal research in view of extrapolating findings from specific animal studies to explain observations done in humans.Materials and Methods AnimalsFor all experiments, naive 15 weeks old male C57Bl/6J mice were used, housed in a temperature and humidity-controlled environment with free access to food and water. Experiments wereCentral NPY and Hepatic VLDL Production in Miceperformed after 4 h of fasting at 12:00 pm with food withdrawn at 8:00 am, unless indicated otherwise. Food intake and body weight were measured weekly during experiments. All animal experiments were approved by the Animal Ethics Committee of the Leiden University Medical Center, Leiden, The Netherlands.Intracerebroventricular SurgeryFor i.c.v. cannula implantation, mice were anaesthetized with 0.5 mg/kg BW Medetomidine (Pfizer, Capelle a/d IJssel, The Netherlands), 5 mg/kg BW Midazolam (Roche, Mijdrecht, The Netherlands) and 0.05 mg/kg BW Fentanyl (Janssen-Cilag, Tilburg, The Netherlands) and placed in a stereotactic device (TSE systems, Homburg, Germany). A 25-gauge guide cannula was implanted into the left lateral ventricle using the following coordinates from Bregma: 1.0 mm lateral, 0.46 mm posterior and 2.2 mm ventral. For third ventricle cannulations the following coordinates from Bregma were used: 0.0 mm lateral, 1.3 mm posterior and 5.7 mm ventral. The guide cannula was secured to the skull surface with dental cement (GC Europe N.V., Leuven, Belgium) and the anesthesia was antagonized using 2.5 mg/kg BW Antipamezol (Pfizer, Capelle a/d IJssel, The Netherlands), 0.5 mg/kg BW Flumazenil (Roche, Mijdrecht, The Netherlands) and 1.2 mg/kg BW Naloxon (Orpha, 1662274 Purkersdorf, Austria). Animals were single housed after the surgery.vehicle (PBS, 100 mL). Both drugs were tested once, in the number of mice indicated. Blood samples were taken from the tail tip into chilled heparin-coated capillaries (Vitrex Medical, Herlev, Denmark) at the indicated time points up to 90 min after tyloxapol injection. The tubes were kept on ice after which they were centrifuged (12.000 rpm for 5 min at 4uC). Plasma TG concentration was determined using a commercially available kit according to the instructions of the manufacturer (no. 11488872, Roche Molecular Biochemicals, Indianapolis, IN) At 120 min, the animals were sacrificed and blood was collected by orbital puncture for isolation of VLDL by density gradient ultracentrifugation [36]. 35S-activity was measured in the VLDL fraction and VLDL-apoB production rate was calculated as dpm.h21 [37].Ve.

D by a 3V injection of NPY (0.2 mg/kg BW) or

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