Neuropeptide Y (NPY), one of the most abundant peptide transmitters in the mammalian brain, is assumed to play an important role in feeding and body weight regulation. However, there is little genetic evidence that overexpression or knockout of the NPY gene leads to altered body weight regulation. Previously, we developed NPY-overexpressing mice by using the Thy-1 promoter, which restricts NPY expression strictly within neurons in the central nervous system, but we failed to observe the obese phenotype in the heterozygote. Here we report that in the homozygous mice, overexpression of NPY leads to an obese phenotype, but only after appropriate dietary exposure. NPY-overexpressing mice exhibited significantly increased body weight gain with transiently increased food intake after 50% sucrose–loaded diet, and later they developed hyperglycemia and hyperinsulinemia without altered glucose excursion during 1 year of our observation period.
Neuropeptide Y (NPY) is a member of the peptide family, which includes the endocrine peptides, pancreatic polypeptide, and peptide YY. A significant body of literature argues that the arcuate nucleus NPY neurons are involved in energy homeostasis (1,2,3,4,5). NPY potently stimulates food intake when introduced into the hypothalamus in the vicinity of the paraventricular nucleus and perifornical area (1,2,3,4,5). NPY alters body temperature and peripheral energy metabolism and increases plasma insulin levels. Many syndromes of obesity in animals are characterized either by an increase in NPY levels in the hypothalamus or by an increase in NPY receptor abundance and sensitivity (3). When NPY was chronically administered into the ventricle or the hypothalamus, it was found to produce an obesity syndrome with salient features of hyperphagia, increased body weight, hyperinsulinemia, hypercorticosteronemia, muscle insulin resistance, and increased fat storage (6). However, because both NPY-overexpressing and knockout mice showed almost normal patterns of food intake and body weight (5,7,8 9), it is still an open question as to whether NPY has an essential role in increased body weight. Here we show that sucrose feeding renders seemingly normal NPY-overexpressing mice to exhibit part of the obesity syndrome.
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 0–24 h and 24–48 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 0–1 h and 1–2 h, respectively, for transgenic versus control mice; n = 9), which was determined with an O2/CO2 metabolism–measuring 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 · dl–1 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).
NPY mediates its effects through binding to the Y-type receptors, and the well-characterized effect on food intake may involve the Y-1 and Y-5 receptor subtypes (5,10). Both receptors are coupled to G proteins and are expressed in the brain, including the hypothalamus. The Y-5 receptor has pharmacological properties most closely matching a proposed feeding receptor (11). However, recent progress in the development of nonpeptide antagonists indicates that the Y-1 receptor mediates the appetite-stimulating effect of NPY (10). In the present study, the levels of Y-1 and Y-5 receptor mRNA expression were found to be affected in the brains of NPY-overexpressing mice, with a significant increase of Y-1 and a decrease of Y-5 receptor expression. Previous studies demonstrated that appetite evoked by either fasting or food restriction increased expression of Y-1 mRNA but did not increase Y-5 mRNA expression (3,12). A significant decrease of Y-5 receptor mRNA expression was found in the ventromedial, dorsomedial, and arcuate hypothalamic nuclei of the obese mouse brain using in situ hybridization (13). Downregulation of the Y-5 receptor in the obese Zucker rat was also demonstrated by competition binding assays (14). These results, together with a significant inhibition of food intake by Y-1 receptor antagonist in the present study, suggest a key role of the Y-1 receptor in the regulation of NPY-induced feeding. It may be possible that the modest overexpression of NPY modifies the balance between Y-1 and Y-5 receptors, resulting in a higher expression of Y-1 receptors.
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 (7,8,9). 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 receptor–deficient 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.
RESEARCH DESIGN AND METHODS
Generation of transgenic mice.
We generated mice (homozygous) that overexpress mouse NPY exclusively in the central nervous system (7). We used a 3.6-kb human Thy-1 DNA spanning the 5′-flanking region containing the first intron (21), which is a major component of the surface of mature neurons in the brain. The DNA fragment (Thy-1-NPY cDNA) was microinjected into the pronuclei of fertilized eggs obtained from BDF1 female mice using standard techniques, as previously reported (7,22).
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 β-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 [α-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 × sodium saline citrate (SSC)/0.1% SDS at room temperature and once for 20 min in 0.1 × 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 β-actin was used as an internal standard.
NPY expression was examined twice (in the 12th and 64th week of each mouse’s 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% sucrose–loaded 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% sucrose–loading in NPY-overexpressing mice. For intra–third cerebroventricular (ICV) injection, the mice (11 weeks of age) were anesthetized with sodium pentobarbital (80–85 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 animal’s 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% sucrose–loaded 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 metabolism–measuring 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.
Northern blot analysis in whole brain of NPY-overexpressing (▪) and control (□) mice. A: Representative response. Expressions of mRNA for NPY, Y-1 receptor (Y-1), and Y-5 receptor (Y-5) were indicated using β-actin as internal standard. B: Changes in mRNA. Statistical significance was determined by t test. Each group contained eight male mice.
Northern blot analysis in whole brain of NPY-overexpressing (▪) and control (□) mice. A: Representative response. Expressions of mRNA for NPY, Y-1 receptor (Y-1), and Y-5 receptor (Y-5) were indicated using β-actin as internal standard. B: Changes in mRNA. Statistical significance was determined by t test. Each group contained eight male mice.
Obesity phenotype of transgenic mice (homozygous). A: Growth curve of NPY-overexpressing (•) and control (○) mice fed on normal diet followed by 50% sucrose–loaded diet. The hatched bars show periods of food intake measurements. B: Cumulative food intake of NPY-overexpressing (▪) and control (□) mice for 3 weeks as indicated in A. C and D: Plasma glucose (C) and insulin (D) levels of transgenic (▪) and control (□) mice. Animals were housed per group under a 12-h light-dark cycle and had free access to food and water. Each group contained 8–10 male mice. *P < 0.05 and **P < 0.01 by t test (A, C, and D only). *P < 0.05 by Mann-Whitney U test; comparison was made on food consumed in each of the four dishes per week (B only).
Obesity phenotype of transgenic mice (homozygous). A: Growth curve of NPY-overexpressing (•) and control (○) mice fed on normal diet followed by 50% sucrose–loaded diet. The hatched bars show periods of food intake measurements. B: Cumulative food intake of NPY-overexpressing (▪) and control (□) mice for 3 weeks as indicated in A. C and D: Plasma glucose (C) and insulin (D) levels of transgenic (▪) and control (□) mice. Animals were housed per group under a 12-h light-dark cycle and had free access to food and water. Each group contained 8–10 male mice. *P < 0.05 and **P < 0.01 by t test (A, C, and D only). *P < 0.05 by Mann-Whitney U test; comparison was made on food consumed in each of the four dishes per week (B only).
Effect of Y-1 receptor antagonist BIBO3304 (2 nmol/mouse every 12 h for 24 h) and Y-5 receptor antagonist L-152,804 (2 nmol/mouse every 12 h for 24 h) on food intake induced by 50% sucrose–loading in NPY-overexpressing mice. **P < 0.01 compared with the control group by analysis of variance followed by Bonferroni’s t test.
Effect of Y-1 receptor antagonist BIBO3304 (2 nmol/mouse every 12 h for 24 h) and Y-5 receptor antagonist L-152,804 (2 nmol/mouse every 12 h for 24 h) on food intake induced by 50% sucrose–loading in NPY-overexpressing mice. **P < 0.01 compared with the control group by analysis of variance followed by Bonferroni’s t test.
Article Information
This study was supported in part by Grants-in-Aid for Scientific Research (09671057), Developmental Scientific Research (08559012), and International Scientific Research (80168418) from the Ministry of Education, Science, Sports, and Culture of Japan (to A.I.).
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.
REFERENCES
Address correspondence and reprint requests to Akio Inui, MD, Associate Professor, Second Department of Internal Medicine, Kobe University School of Medicine, 7-5-2 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan. E-mail: [email protected].
Received for publication 16 March 2000 and accepted in revised form 16 January 2001.
AUC, area under curve; ICV, intra–third cerebroventricular; NPY, neuropeptide Y; SSC, sodium saline citrate.
