1 Second Department of Internal Medicine, Kobe University School of Medicine, Kobe
2 Department of Anatomy, Shiga University of Medical School, Otsu
3 Research & Development Division, Nippon Organon K.K.
4 Kampo & Healthcare Research Laboratories, Kanebo, Osaka, Japan
5 Department of Pharmacology, College of Medicine, National Cheng Kung University, Tainan, Taiwan, China
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ABSTRACT |
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INTRODUCTION |
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NPY-overexpressing mice (homozygous) were developed by using the Thy-1 promoter, which localized transgene expression within the central nervous system (7). A modest overexpression of NPY was confirmed in these animals (Fig. 1), which were housed per group and examined in a less stressful condition than those housed individually. There were no apparent differences in body weight between NPY-overexpressing mice and their littermates when normal diet was provided. However, shortly after 50% sucrose was loaded on the diet, the homozygous mice became heavier than controls and maintained their increased body weight (Fig. 2A). This was accompanied by an increase in food intake that continued for several weeks after the high-sucrose feeding (Fig. 2B). The increased food intake in transgenic mice was significantly inhibited by the treatment with the NPY Y-1 receptor antagonist BIBO3304, but not the Y-5 receptor antagonist L-152,804 (Fig. 3). There was a significant difference in energetic efficiency, shown by a smaller decrease in body weight in response to 48-h fasting observed in transgenic mice compared with the control mice (13.4 ± 0.34 vs. 13.1 ± 0.96 and 8.11 ± 0.33 vs. 9.31 ± 0.41% body wt change at 024 h and 2448 h, respectively; P < 0.05, n = 7). The mice did not show any change in oxygen consumption (7.07 ± 0.31 vs. 7.38 ± 0.39 and 5.33 ± 0.24 vs. 5.62 ± 0.38 ml/g body wt/h at 01 h and 12 h, respectively, for transgenic versus control mice; n = 9), which was determined with an O2/CO2 metabolismmeasuring system at 25°C. NPY-overexpressing mice showed increased plasma levels of glucose and insulin (Figs. 1C and D), although they did not show impaired glucose tolerance after per os glucose loading (2 g/kg, area under curve [AUC] for glucose 369.0 ± 19.6 vs. 393.6 ± 35.8 mg · h · dl1 for transgenic versus control mice; n = 9). The levels of total cholesterol and triglycerides did not differ appreciably between transgenic and control animals (not shown).
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Very recently, transgenic rats were generated by using the NPY genomic sequence (15). They exhibited behavioral insensitivity to stress and fear and impaired spatial learning, but they exhibited neither feeding nor body weight abnormalities, presumably because of the limited overexpression of NPY to areas outside those controlling feeding behaviors (15). Energy balance could be well maintained when NPY was removed or mildly overexpressed from early development of the nervous system (789). Moreover, mutant mice that lacked either the Y-1 or Y-5 receptor paradoxically developed obesity, despite the decrease in the effects of NPY and analogs on food intake (16,17). The unexpected phenotype of Y-1 and Y-5 knockouts developed later and was associated with decreased locomotor activity and increased food intake, respectively. In contrast, a recent study indicated an inhibitory role for the Y-2 receptor in the control of food intake and body weight. The Y-2 receptordeficient mice developed obesity, especially when fed a high-fat diet (14). This may be in keeping with a presynaptic function for the Y-2 receptor in the hypothalamus (i.e., an inhibition of NPY release) (18), although Y-2 receptor agonists have the least effect on feeding when injected into the cerebral ventricle (10). As a whole, these studies present a complex picture for the role of NPY and its receptors in feeding and body weight regulation.
The present study provides direct genetic evidence that overexpression of NPY leads to obesity in the presence of a palatable diet, with increased food intake and energetic efficiency. The obesity phenotype in transgenic animals may be critically dependent on environmental (dietary) exposure. Previous studies in diet-induced obesity models suggested that there may be some central "set point" for the regulation of body weight that is genetically predetermined but expressed only in the presence of appropriate dietary exposure (19,20). Altered NPY regulation in the hypothalamus may be a characteristic feature, and the NPY system can respond both to the metabolic needs of the animals and the palatability of the food (20). Our model may thus be particularly useful for the study of diet-induced obesity, which is seen in most human obesity and diabetes.
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RESEARCH DESIGN AND METHODS |
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Northern blot analysis.
Total RNA was extracted from the whole brain of experimental animals using the Ultraspec-II RNA extraction system (Bioteck, Houston, TX). The concentration of RNA was measured using the absorbance at 260 nm. For Northern blot analysis, RNA (20 µg) was denatured in a solution containing 2.2 mmol/l formaldehyde and 50% formamide (vol/vol) by heating at 55°C for 15 min. Aliquots of total RNA were then size-fractionated in a 1.2% agarose/formaldehyde gel (23). Ethidium bromide staining was used to identify the position of the 18S and 28S rRNA subunits and to confirm that equivalent amounts of undegraded RNA had been loaded. The fractionated RNA was transferred to a Hybond-N membrane (Amersham, Bucks, U.K.) and crosslinked by UV irradiation (Stratagene, La Jolla, CA).
The plasmid containing cDNA for NPY was supplied by Dr. Steven L. Sabol (National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD). The plasmid containing cDNA of Y-1 and Y-5 was obtained from Dr. Herbert Herzog (The Garvan Institute of Medical Research, Sydney, Australia), and the ß-actin cDNA was from Dr. S.S. Liu (Department of Microbiology and Immunology, National Cheng Kung University, Taiwan City, Taiwan, China). Probes were labeled with [-32P]dCTP using the Megaprime labeling system kit (Amersham). Hybridizations were carried out in medium containing denatured salmon sperm DNA (100 µg/ml) at 65°C for 2 h. The membrane was washed twice for 20 min in 2 x sodium saline citrate (SSC)/0.1% SDS at room temperature and once for 20 min in 0.1 x SSC/0.1% SDS at 40°C. Autoradiograms were prepared on Kodak X-ray film (Rochester, NY) using a single enhancing screen at 70°C (laid down for 16 h). Intensity of the mRNA bands on the blot was quantified by scanning densitometry (Hoefer, San Francisco, CA). The response of ß-actin was used as an internal standard.
NPY expression was examined twice (in the 12th and 64th week of each mouses life), and the animals were killed at 10:00 A.M. by cervical dislocation. Essentially, the same results were obtained in each study and the latter data were presented.
Behavioral analysis.
Transgenic mice (male) were housed per group in a regulated environment (22 ± 2°C, 55 ± 10% humidity, and 12-h light-dark cycle). The cumulative food intake of NPY-overexpressing and control mice was measured every 3 weeks before and after the start of 50% sucroseloaded powder diet. Experiments were performed beyond 1 year.
NPY receptor antagonist experiments.
We examined the effect of the selective NPY receptor antagonists on food intake induced by 50% sucroseloading in NPY-overexpressing mice. For intrathird cerebroventricular (ICV) injection, the mice (11 weeks of age) were anesthetized with sodium pentobarbital (8085 mg/kg i.p.) and placed in a stereotaxic instrument (SR-6; Narishige, Tokyo) 7 days before the experiments. A hole was made in each skull by using a needle inserted 0.9 mm lateral to the central suture and 0.9 mm posterior to the bregma. A 24-gauge cannula beveled at one end over a distance of 3 mm (Safelet-Cas; Nipro, Osaka, Japan) was implanted into the third cerebral ventricle for ICV injection. The cannula was fixed to the skull with dental cement and capped with silicon without an obtruder. A 27-gauge injection insert was attached to a microsyringe by PE-20 tubing. Using tweezers, this could easily be inserted into a fixed cannula without holding the mouse and thus without greatly disturbing the animals behavior. After completion of the experiment, the location of the cannula tip was confirmed by dye injection (Evans blue 0.5% and Zelatin 5%) and histological examination of frozen brain sections (24).
NPY-overexpressing and control mice at 12 weeks of age were fed on 50% sucroseloaded powder diet after implantation. Cumulative food intake was measured before and after ICV injection of NPY Y-1 and Y-5 receptor antagonists. BIBO3304 was supplied by Boeringer-Ingelheim Pharma (Grenzach-wyhlen, Germany), and L-152,804 was from Banyu Pharmaceutical (Tokyo). Just before administration, each drug was diluted in 4 µl of artificial cerebrospinal fluid, which also served as the control solution, and ICV was administered at a dose of 2 nmol/mouse every 12 h. The dosage of the antagonists was chosen based on our previous study (25) to rule out potential nonspecific effects.
Energy expenditure.
Oxygen consumption was determined with an O2/CO2 metabolismmeasuring system (model MK-5,000; Muromachikikai, Tokyo) at 25°C in nine transgenic and nine control mice. The chamber volume was 560 ml, and airflow to the chamber was 500 ml/min; samples were taken every 3 min, and a standard gas reference was taken every 30 min. Mice were kept unrestrained in the chamber without food or water during the light cycle, and oxygen consumption was measured for 2 h. Essentially, the same results were obtained in the 12th and 60th weeks of the animals lives, and the latter data were presented.
Glucose tolerance.
Plasma glucose and insulin were measured four times in nonfasting conditions. Oral glucose tolerance (2 g/kg body wt) was performed at 64 weeks, and AUC for glucose was determined. Plasma glucose and insulin were determined by Glucose B test (Wako, Osaka, Japan) and enzyme-linked immunosorbent assay insulin kit (Morinaga, Tokyo), respectively.
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ACKNOWLEDGMENTS |
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The authors sincerely thank Dr. Steven L. Sabol (National Heart, Lung, and Blood Institute, National Institutes of Health) and Dr. Herbert Herzog (The Garvan Institute of Medical Research) for kindly supplying us the plasmid containing cDNA for NPY and for Y-1 and Y-5 receptors, respectively.
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FOOTNOTES |
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Received for publication 16 March 2000 and accepted in revised form 16 January 2001.
AUC, area under curve; ICV, intrathird cerebroventricular; NPY, neuropeptide Y; SSC, sodium saline citrate.
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REFERENCES |
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