Y2Y4 Receptor Double Knockout Protects Against Obesity Due to a High-Fat Diet or Y1 Receptor Deficiency in Mice
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糖尿病学杂志 2006年第1期
1 Neurobiology Research Program, The Garvan Institute of Medical Research, St. Vincent’s Hospital, Sydney, Australia
2 Diabetes Research Program, The Garvan Institute of Medical Research, St. Vincent’s Hospital, Sydney, Australia
3 Department of Pharmacology and Toxicology, University of Innsbruck, Innsbruck, Austria
4 Neuroscience Institute of Schizophrenia and Allied Disorders, Sydney, Australia
5 Bone and Mineral Research Program, The Garvan Institute of Medical Research, St. Vincent’s Hospital, Sydney, Australia
6 Department of Pharmacology, Innsbruck Medical University, Innsbruck, Austria
POMC, proopiomelanocortin; WATe, epididymal white adipose tissue
ABSTRACT
Neuropeptide Y receptors are critical regulators of energy homeostasis, but the functional interactions and relative contributions of Y receptors and the environment in this process are unknown. We measured the effects of an ad libitum diet of normal or high-fat food on energy balance in mice with single, double, or triple deficiencies of Y1, Y2, or Y4 receptors. Whereas wild-type mice developed diet-induced obesity, Y2Y4 double knockouts did not. In contrast, Y1 knockout or Y1Y2 or Y1Y4 receptor double knockout mice developed an exacerbated diet-induced obesity syndrome. Remarkably, the antiobesity effect of Y2Y4 deficiency was stronger than the obesogenic effect of Y1 deficiency, since Y1Y2Y4 triple knockouts did not develop obesity on the high-fat diet. Resistance to diet-induced obesity in Y2Y4 knockouts was associated with reduced food intake and improved glucose tolerance in the absence of changes in total physical activity. Fecal concentration of free fatty acids was significantly increased in Y2Y4 knockouts in association with a significantly reduced bile acid pool and marked alterations in intestinal morphology. In addition, hypothalamic proopiomelanocortin expression was decreased in diet-induced obesity (in both wild-type and Y1 receptor knockout mice) but not in obesity-resistant Y2Y4 receptor knockout mice fed a high-fat diet. Therefore, deletion of Y2 and Y4 receptors synergistically protects against diet-induced obesity, at least partially via changes in food intake and hypothalamic proopiomelanocortin expression.
Members of the Y receptor family, notably Y1, Y2, Y4, and Y5 receptors, are implicated in energy homeostasis and the development of obesity and insulin resistance. These receptors are activated by three endogenous ligands: neuropeptide Y, the gut-derived hormones peptide YY, and pancreatic polypeptide. Recently, there has been renewed speculation that ligands for Y receptors, such as peptide YY3eC36 and pancreatic polypeptide, may be of benefit for the treatment of obesity (1).
There is considerable conflict in the literature about the role of Y receptors in the regulation of body weight. For instance, pharmacological studies suggested that Y1 receptors contribute to hyperphagia induced by increased hypothalamic neuropeptide Y secretion (2). Fasting-induced refeeding is reduced in Y1 knockout mice (3). Moreover, food intake and body weight of genetically obese ob/ob mice, in which hypothalamic neuropeptide Y-ergic activity is chronically increased, are significantly reduced by Y1 knockout (4). In contrast, Y1 knockouts develop significant increases in body weight, fat mass, and insulinemia in the absence of hyperphagia (3,5).
Similar controversies prevail regarding the role of Y2 receptors in energy homeostasis. Hypothalamus-specific or germ-line deletion of Y2 receptors resulted in significant reductions in the body weight of lean mice (6) and significant reductions in adiposity or body weight and the type 2 diabetic syndrome of ob/ob mice in the absence of reductions in food intake (7,8). In contrast, another germ-line Y2 receptor knockout model was shown to develop increased body weight, fat deposition, and hyperphagia (9). Furthermore, intrahypothalamic administration of the Y2 agonist neuropeptide Y[13eC36] to rats significantly decreased food intake (10). Moreover, circulating peptide YY from the gut and its truncated form peptide YY3eC36 (a preferential Y2 agonist) have been shown to reduce food intake, adiposity, and/or body weight after short- or long-term administration to lean or obese rodents or humans (1,11,12).
Recent work suggests that pancreatic polypeptide, a preferential Y4 receptor agonist secreted by the F cells of the islets of Langerhans, regulates energy homeostasis. Intravenous or intraperitoneal administration of pancreatic polypeptide to mice increases metabolic rate (13) and decreases hyperglycemia, basal or glucose-induced hyperinsulinemia, insulin resistance, and hyperlipidemia in ob/ob mice, culminating in decreases in adiposity and body weight (13,14). Pancreatic polypeptide transgenic mice, which have a 20-fold increase in plasma pancreatic polypeptide levels, have reduced food intake, body weight, and fat mass as well as reduced glucose-induced insulin secretion and gastric emptying when compared with control mice (15). However, other findings do not support a role of Y4 receptors in energy homeostasis. Y4 receptor knockout had no effect on the hyperphagia, obesity, or type 2 diabetic phenotype of ob/ob mice (16).
These conflicting data make it difficult to define the true physiological and pathophysiological role of Y receptors in energy homeostasis and obesity. Moreover, it is difficult to know whether potential obesity treatments targeted to Y receptors should act as agonists or antagonists. Part of this conflict can be explained by the fact that Y receptors mediate their physiological effects via complex synergistic and redundant interactions. For instance, knockout of individual Y receptors alters ligand-binding patterns of the remaining Y receptors (17). In addition, ligands for Y receptors are renowned for their lack of specificity for individual receptor types.
To circumvent some of these complications of Y receptor biology, we made a systematic study of energy balance in mice with single, double, or triple knockout of Y1, Y2, or Y4 receptors, both under chow-fed conditions and in diet-induced obesity. In this way, we were able to clarify the interactions among these receptor types in regulating energy balance (including body weight, food intake, physical activity, thermogenesis, glucose tolerance, intestinal lipid absorption, intestinal morphology, and hypothalamic expression of neuropeptide Y and proopiomelanocortin) and to elucidate their roles in the development of diet-induced obesity. Y5 receptor knockouts were not investigated in this study because the Y5 and Y1 receptors are localized only 20 kb apart on the same chromosome, and it is therefore not plausible to obtain Y1Y5 receptor double knockout mice by cross-breeding.
RESEARCH DESIGN AND METHODS
Generation of the Y1, Y2, and Y4 knockout mice were previously published (6,16,18). Male double or triple knockout mice were obtained by crossing Y1, Y2, or Y4 knockout mice, respectively. All mice were on a mixed C57BL/6-129/SvJ background. All research and animal care procedures were approved by the Garvan Institute/St. Vincent’s Hospital Animal Experimentation Ethics Committee and were in agreement with the Australian Code of Practice for the Care and Use of Animals for Scientific Purpose. Mice were housed under conditions of controlled temperature (22°C) and illumination (12-h light cycle, lights on at 0700). Half of the mice of each genotype were fed a normal chow diet ad libitum (6% calories from fat, 21% calories from protein, 71% calories from carbohydrate, and 2.6 kcal/g; Gordon’s Speciality Stock Feeds, Yanderra, NSW, Australia). The other half was fed ad libitum with a high-fat diet supplemented with fat and sucrose (46% calories from fat, 21% calories from protein, 33% calories from carbohydrate, and 4.72 kcal/g) from 8 weeks of age onwards. The diet was based on the composition of rodent diet catalog no. D12451 (Research Diets, New Brunswick, NJ), with the exception that safflower oil and copha were used in place of soybean oil and lard. Twenty-four-hour food intake was estimated twice a week for 4 weeks from 13 to 16 weeks of age by the average amount of food removed from the food hopper. In a subset of animals, the actual proportion of food that was consumed was calculated by subtracting the weight of the food spilt in the cage from the weight of food removed from the hopper. Body temperature was determined at 14eC16 weeks of age (BAT-10 multipurpose thermometer; Physitemp Instruments, Clifton, NJ).
Determination of physical activity.
Distance traveled in the open field test was determined as previously described (19). Total cage activity was recorded using a passive infrared detector (Conrad Electronics, Hanover, Germany) on top of the cage lid of single-housed animals. The signals of the passive infrared detectors were detected by an I/O interface card (PIO48 II; Conrad Electronics) and stored using custom-made software. A mean activity per day was calculated by averaging corresponding 30-min intervals of 7 consecutive days.
Glucose tolerance tests and tissue collection.
At 16 weeks of age, animals were fasted overnight and then administered an intraperitoneal injection of D-glucose (60 mg/mouse). Glucose levels were determined in tail blood samples with glucose test strips (Accu-Check Advantage II Glucose Test Strips; Roche Diagnostics Australia, Castle Hill, NSW, Australia). At 3eC8 days after the glucose tolerance test, food was removed from cage hoppers at 0900, and animals were killed 4eC7 h later by cervical dislocation followed by cardiac puncture for serum collection. Brains were immediately removed and frozen on dry ice. The interscapular brown adipose tissue was removed, weighed, and stored frozen until analysis for uncoupling protein-1 mRNA levels (20). Epididymal white adipose tissue (WATe) depots were dissected and weighed. The small intestine of some mice was removed and prepared for histology as described below. In separate groups of mice, the small intestine was flushed with isotonic saline, blotted on paper towels, and weighed. In other mice, the liver, gallbladder, small intestine and contents were removed, weighed, and stored at eC80°C until analysis of total bile acid pool (21).
Analysis of fecal lipids and bile acids.
Fecal samples were dried to a constant weight at 90°C. Some fecal samples were used for extraction of total lipids (22). Lipids were resuspended in pure chloroform and separated by TLC (thin-layer chromatography) on Whatman Silica Gel K6 glass TLC plates (Whatman, Maidstone, England) in hexane:diethyl ether:acetic acid, 80:20:1. Standards were triolein (50 e蘥), dipalmitine (75 e蘥), and palmitine (125 e蘥) (Sigma, St. Louis, MO). Fractions corresponding to triglycerides, diglycerides, monoglycerides, and free fatty acids were scraped from the plate for assay of triacylglycerol (23). The free fatty acid fraction was resuspended in chloroform and assayed for free fatty acids with a colorimetric assay kit (WAKO, Saitama, Japan). Other samples of feces were used for determination of fecal bile acid concentration (21).
Analysis of intestinal morphology.
One-centimeter pieces of small intestine were taken from the following three locations: duodenum, 3 cm distal from the stomach-duodenal junction; jejunum, at the midpoint of the small intestine; and ileum, 3 cm proximal from the ileocaecal junction. Intestinal pieces were fixed for 16eC18 h in Bouin’s solution (Sigma), embedded in paraffin, sectioned at 7 e蘭, mounted on charged slides (SuperFrost Plus; Menzel-Glaser, Braunschweig, Germany), and stained with periodic acid Schiff. Villus length (from base of the crypt to tip of the villus) was measured in at least 15 villi from three mice in each group, using a light microscope (Axiophot; Zeiss, Oberkochen, Germany) connected to a Leica camera and Leica Image Manager software (Leica, Heerbrugg, Switzerland). The number of goblet cells per villus (excluding crypts) were counted in at least 15 villi from five mice in each group. All analyses were done by investigators unaware of genotype.
Neurochemical analyses.
Coronal brain slices (20 e蘭) at the level of the hypothalamic arcuate nucleus were hybridized using DNA oligonucleotides complementary to mouse neuropeptide Y and proopiomelanocortin (POMC) (7).
Serum analyses.
Serum hormone levels were determined with commercial radioimmunoassay kits from Linco Research (St. Louis, MO) (leptin and insulin), ICN Biomedicals (Costa Mesa, CA) (corticosterone, free T4, and testosterone), and Bioclone Australia (Marrickville, Australia) (IGF-1). Basal serum glucose levels were determined with a glucose oxidase kit (Trace Scientific, Melbourne, Australia).
Statistical analysis.
All data are expressed as means ± SE. Differences among groups of mice were assessed by ANOVA or repeated-measures ANOVA, followed by Fisher’s post hoc comparisons, if appropriate (StatView version 4.51; Abacus Concepts, Berkeley, CA). Statistical significance was defined as P < 0.05.
RESULTS
Effects of Y receptor deficiency on diet-induced obesity.
The body weights of mice at 8 weeks of age (before the introduction of the high-fat diet) were not significantly different from each other, except for Y1Y2Y4 triple knockout mice, which were lighter (wild-type, 23.7 ± 0.3 g; Y1, 22.8 ± 0.6 g; Y1Y2, 24.0 ± 0.4 g; Y1Y4, 24.2 ± 0.4 g; Y2, 24.5 ± 0.6 g; Y4, 24.4 ± 0.3 g; Y2Y4, 24.8 ± 0.4 g; and Y1Y2Y4, 22.1 ± 0.5 g, P < 0.05 vs. wild-type, n = at least 11 mice per genotype). Figure 1A, shows that under chow-fed conditions, body weight gain from 8 weeks until 16 weeks of age was similar in all groups of mice, except for Y1 knockout and Y1Y4 double knockout mice. The absolute and relative weights of the WATe depot and serum leptin levels were similar in all groups of mice under chow-fed conditions, except for Y2Y4 double and Y1Y2Y4 triple knockout mice, which showed significant decreases (Fig. 1B and C, Fig. 2A, ). Under chow-fed conditions, serum insulin levels were significantly increased over wild-type values in Y1 and Y1Y4 double knockout mice, and significantly decreased relative to wild-type in Y2Y4 double knockouts (Fig. 2B, ). In chow-fed animals, there were no significant differences among groups with respect to serum levels of corticosterone or free T4 (Fig. 1C and D), glucose, testosterone, or IGF-1 (data not shown). When fed ad libitum for 8 weeks on the high-fat diet, wild-type mice developed a distinct obesity syndrome characterized by increased body weight gain (Fig. 1A), increased WATe weight (Fig. 1B and C), and increases in serum leptin, insulin, and corticosterone levels (Fig. 2AeCC), with a tendency to decreased free T4 levels (Fig. 2D). Y1 receptor deficiency, either alone or in combination with Y2 or Y4 receptor deficiency (i.e., Y1, Y1Y2, and Y1Y4 receptor knockout mice), exacerbated this diet-induced obesity syndrome (Figs. 1AeCC and 2B). Y1, Y1Y2, and Y1Y4 receptor knockout mice also developed hyperleptinemia, hypercorticosteronemia, and a tendency to decreased free T4 levels after high-fat feeding, but these responses were not significantly different from wild-type animals (Fig. 2A,C,D). In contrast to the exacerbating effect of Y1 deficiency on diet-induced obesity, Y2 or Y4 deletion (in Y2, Y4, Y2Y4 double, and Y1Y2Y4 triple knockout mice) conferred protection against diet-induced obesity (Figs. 1 and 2). However, similar to wild-type mice, Y2, Y2Y4 double, and Y1Y2Y4 triple knockout mice showed marked decreases in serum free T4 levels in response to high-fat feeding (Fig. 2D).
Effects of Y receptor deficiency on feeding behavior and food intake.
As shown by the open columns in Fig. 3, Y1Y4, Y4, Y2Y4 double, and Y1Y2Y4 triple knockout mice removed significantly more normal chow from the hopper everyday compared with wild-type mice. When fed the high-fat diet, Y1Y4, Y2Y4 double, and Y1Y2Y4 triple knockout mice removed significantly more high-fat diet from the cage hopper than wild-type controls (Fig. 3, ). Y2Y4 double knockout mice spilled significantly more of the food taken from the cage hopper than wild-type mice, and the amount of chow or high-fat food actually consumed was significantly less than that consumed by wild-types (Fig. 4). There was no significant effect of diet or genotype on water intake (data not shown). Because double deletion of Y2 and Y4 receptors conferred resistance against diet-induced obesity (even in combination with Y1 receptor deficiency), we focused our attention from here onwards on the Y2Y4 knockouts.
No effect of Y2Y4 receptor double knockout on physical activity or thermogenesis.
In the open field test, there was no significant difference between Y2Y4 double knockout mice and wild-type controls with respect to total distance traveled (6,960 ± 510 vs. 5,830 ± 240 cm/30 min, respectively; n = at least 4 mice per group). Moreover, there was no significant difference between double knockout and wild-type mice with respect to total physical activity in the home cage (data not shown).
As an index of nonactivity thermogenic capacity, we measured expression of uncoupling protein-1 mRNA in brown adipose tissue (24) and rectal temperature in Y2Y4 double knockout and wild-type mice. There was no significant difference between groups with respect to uncoupling protein-1 mRNA levels in brown adipose tissue, standardized to glyceraldehyde-3-phosphate dehydrogenase mRNA levels and expressed as a percent of wild-type values (107.3 ± 6.7 vs. 100.0 ± 7.4% in Y2Y4 double knockout and wild-type mice, respectively; n = 5 mice per group). Moreover, rectal temperature of chow- or fat-fed Y2Y4 receptor double knockout mice was not significantly different from that of wild-type mice fed on the same diet (data not shown).
Enhanced glucose tolerance in Y2Y4 receptor double knockout mice.
There was no significant difference among groups with respect to peak glycemia following intraperitoneal glucose injection. However, in Y2, Y2Y4 double, and Y1Y2Y4 triple knockout mice, there was a significant reduction in serum glucose levels at 60 and 90 min postinjection compared with wild-type mice (Fig. 5). All other groups of mice (Y1, Y1Y2 double, Y1Y4 double, and Y4 knockout mice) showed glucose clearance curves indistinguishable from wild-type mice (Fig. 5).
Decreased intestinal lipid absorption in Y2Y4 receptor double knockout mice.
Fig. 6 shows the concentration of triglycerides, diglycerides, and free fatty acids in feces from wild-type and Y2Y4 receptor double knockout mice, either under chow-fed (Fig. 6A) or high-fateCfed (Fig. 6B) conditions. In both groups of mice, free fatty acids were the most abundant fecal lipid species, and undigested or partially digested lipids (triglycerides and diglycerides) were 6- to 30-fold less abundant. Monoglycerides were not detectable in the fecal samples investigated. These findings indicate that triglyceride digestion to free fatty acids proceeds efficiently in both wild-type and Y2Y4 receptor double knockout mice. However, absorption of digested lipids may be impaired in Y2Y4 double knockout mice, since the fecal concentration of free fatty acids was increased in knockout versus wild-type mice, significantly so during high-fat feeding (Fig. 6B). Daily fecal output (dried weight) from Y2Y4 double knockout mice on a chow diet was significantly less than that of wild-type controls (0.64 ± 0.12 vs. 1.23 ± 0.04 g · mouseeC1 · dayeC1, n = 3eC4 cages of 2eC4 mice per group). However, there was no significant difference in daily fecal output between knockout and wild-type mice on the high-fat diet (0.22 ± 0.02 vs. 0.25 ± 0.04 g · mouseeC1 · dayeC1 in wild-types, n = 3eC4 cages of 2eC4 mice per group).
Reduced bile pool and differences in intestinal morphology in Y2Y4 receptor double knockout mice.
We determined the total bile pool because intestinal lipid absorption is dependent on bile production. The total bile pool of Y2Y4 knockouts was significantly reduced compared with wild-type mice (20.0 ± 3.6 vs. 41.0 ± 3.0 e蘭ol total bile, respectively; and 88 ± 17 vs. 142 ± 10 e蘭ol/100 g body wt, respectively; means ± SE of 12eC13 mice per group, P < 0.01). Fecal total bile acid concentration was similar in Y2Y4 knockout and wild-type mice (33 ± 9 vs. 34 ± 2 e蘭ol/g dried feces, respectively, means ± SE of at least three mice per group). Since daily fecal output was reduced by 50% in Y2Y4 versus wild-type mice, total bile acid output of Y2Y4 knockouts was therefore half that of wild-type counterparts, in keeping with the smaller total bile acid pool size.
Although there was no significant difference between wild-type and knockout mice with regards to either weight (1.1 ± 0.05 vs. 1.1 ± 0.05 g, respectively; means ± SE of 22eC24 mice per group) or length (39.4 ± 0.8 vs. 37.6 ± 0.6 cm, respectively; means ± SE of 25eC27 mice per group) of the small intestine, the average length of small intestinal villi was significantly decreased in the duodenum of Y2Y4 double knockout versus wild-type mice (431 ± 2 vs. 525 ± 6 e蘭, respectively, n = 3 mice per genotype, P < 0.05) (Fig. 7A and B). This difference in villus length was not observed in the jejunum (295 ± 16 vs. 287 ± 6 e蘭; Y2Y4 and wild-type mice, respectively, n = 3 mice per genotype) or ileum (204 ± 24 vs. 184 ± 15 e蘭 in Y2Y4 and wild-type mice, respectively, n = 3 mice per genotype). Y2Y4 receptor double knockout mice had an increase over wild-type mice in the number of goblet cells in the duodenum (23.7 ± 1.7 vs. 20.6 ± 1.0 goblet cells per villus in wild-type mice, n = 5 mice per group, NS), the jejunum (21.6 ± 1.1 vs. 18.0 ± 0.6 goblet cells per villus in wild-type mice, n = 5 mice per group, P < 0.05), and the ileum (23.1 ± 1.4 vs. 18.2 ± 0.6 goblet cells per villus in wild-type mice, n = 5 mice per group, P < 0.05). Representative villi from the ileum of wild-type and Y2Y4 receptor knockout mice are shown in Fig. 7C and D.
Altered hypothalamic expression of neuropeptide Y and POMC in Y receptor knockout mice.
In wild-type mice, the high-fat diet resulted in significant decreases in neuropeptide Y and POMC mRNA levels in the arcuate nucleus (Table 1). Increased weight gain in both chow-fed and high-fateCfed Y1 receptor knockout mice, compared with wild-types, was associated with decreased neuropeptide Y and POMC mRNA levels compared with wild-type values (Table 1). In addition, in Y1 receptor knockout mice, diet-induced obesity was associated with decreased neuropeptide Y and POMC mRNA levels compared with chow-fed mice (Table 1). In contrast, in Y2Y4 receptor knockout mice, neuropeptide Y and POMC mRNA levels were not significantly decreased following ingestion of a high-fat diet compared with chow-fed double knockouts. Rather, neuropeptide Y and POMC mRNA levels were increased in Y2Y4 receptor knockout mice compared with wild-type mice fed the same diet (Table 1).
DISCUSSION
These data demonstrate that ablation of Y2 and Y4 receptors protects against diet-induced obesity, probably via reduced food intake. In contrast, deletion of Y1 receptors significantly exacerbates diet-induced obesity. Moreover, the antiobesity effect of double Y2 and Y4 receptor deficiency overrides the obesogenic effect of Y1 receptor deficiency and a high-fat diet (as seen in Y1Y2Y4 triple knockout mice). These observations provide evidence that the molecular system that regulates energy homeostasis, including the Y receptor system, has less redundancy and capacity for compensation to favor positive over negative energy balance than previously thought.
We recently showed that Y2 and Y4 receptor knockout synergistically increases bone volume in conjunction with a significant increase in bone formation rate (25). It is therefore conceivable that in addition to decreased food intake, this threefold increase in bone volume could contribute to the lean phenotype observed in Y2Y4 receptor double knockout mice.
The current work suggests that Y2Y4 receptor double deficiency decreases absorption of fat from the digestive tract. This change was not associated with any decrease in efficiency of triglyceride digestion to free fatty acids. The reduced intestinal fat absorption in Y2Y4 receptor knockout mice is likely to be mediated, at least in part, by the reduced total bile pool observed in these animals. Indeed, fat absorption and energy extraction from food is positively correlated with the concentration of functional bile acids (26,27). It is unlikely however that decreased intestinal lipid absorption contributes to the lean phenotype of Y2Y4 receptor knockout mice, since the calorie content of the extra free fatty acids excreted in the feces of these mice represents <1% their total daily calorie intake. Previous work has shown that both peptide YY and pancreatic polypeptide inhibit gallbladder emptying (28) or gallbladder bile output in pigs and/or humans (28,29), presumably via interactions with Y receptors in the dorsovagal complex of the brain stem and subsequent vagal-dependent pathways (28,30). The current work extends these data to show the unambiguous involvement of Y2 and Y4 receptors in the regulation of total bile pool.
Reduced intestinal absorption of lipids in Y2Y4 receptor double knockout mice may be influenced by the observed decrease in villus length of the duodenum. It is also possible that the increased number of goblet cells observed in the small intestine of Y2Y4 receptor double knockout mice may have led to increased production of intestinal mucus, which could accelerate intestinal transit and impair absorption of nutrients (31).
In contrast to the protective effects of Y2 and Y4 receptor deficiency against diet-induced obesity, animals deficient in Y1 receptors had a tendency for increased adiposity under chow-fed conditions and had an exacerbated obesity syndrome in response to the high-fat diet. This was observed in single Y1 receptor knockout animals as well as in Y1Y2 and Y1Y4 double knockout animals. In Y1Y2Y4 receptor triple knockout animals however, the antiobesity effect of Y2 and Y4 receptor deficiency predominated over the obesogenic effect of Y1 deficiency and a high-fat diet. It is likely that hormonal and metabolic alterations, notably hyperinsulinemia, contribute to the increased propensity for obesity in Y1, Y1Y2, and Y1Y4 receptor knockout mice. When injected peripherally or when administered to pancreatic tissue in vitro, neuropeptide Y decreases insulin secretion (32,33), probably via direct interaction with the Y1 receptors expressed on pancreatic -cells (34,35). It is likely that the lack of Y1 receptors on pancreatic islet tissue contributed to the increased basal serum insulin levels of Y1, Y1Y2, and Y1Y4 receptor knockout mice, with hyperinsulinemia subsequently contributing to increased adiposity (36). Indeed, we have shown that glucose-induced serum insulin levels are increased in Y1 knockout compared with wild-type mice (A.S., N.J.L., K.S., R.E., and H.H., unpublished data). Other groups have also reported that germline Y1 receptor knockout induces obesity and basal hyperinsulinemia (3,5,37) and that hyperinsulinemia is likely to be a key etiological feature in the obese phenotype of these mice (37).
High-fat feeding resulted in decreased expression of neuropeptide Y in the arcuate nucleus of wild-type mice and Y1 receptor knockout mice, possibly as an adaptive response to inhibit further weight gain. This is most likely mediated via increased circulating concentrations of leptin and the gut peptide peptide YY3eC36, release of which is strongly increased by dietary fat (38,39), subsequently acting on leptin or Y2 receptors in the arcuate nucleus to reduce neuropeptide Y expression levels and thereby reducing feeding (1,40,41). Expression of POMC in the arcuate nucleus of wild-type and Y1 receptor knockout mice fed a high-fat diet was also reduced, a change that may contribute to the diet-induced obesity observed in these mice by decreasing secretion of -melanocyte stimulating hormone. Consistent with this hypothesis, rodent obesity models of genetic (42,43) or dietary (44) origin exhibit decreased expression of POMC in the arcuate nucleus. It was of interest that the decreases in hypothalamic expression of neuropeptide Y and POMC induced by the high-fat diet were absent in obesity-resistant Y2Y4 receptor knockout mice and were exacerbated in obesity-prone Y1 receptor knockout mice. These findings suggest a possible causative role of changes in POMC signaling in the development or resistance to diet-induced obesity in wild-type, Y1 knockout, and Y2Y4 receptor double knockout mice.
We recently demonstrated that single, double, or triple deletion of Y1, Y2, or Y4 receptors alters the expression or binding patterns of the remaining Y receptors in the brain (17). However, the major changes observed are located in areas not likely to participate in the regulation of energy homeostasis, notably the hippocampus. Therefore, it is unlikely that the changes in energy homeostasis observed in our panel of Y receptor knockout mice were due to compensatory changes in other Y receptors remaining in the hypothalamus.
In conclusion, these data demonstrate that Y2 and Y4 receptors are necessary for diet-induced obesity, at least in part via effects on food intake and hypothalamic POMC expression. In contrast, Y1 receptors inhibit diet-induced obesity, probably via direct inhibition of insulin secretion from pancreatic -cells. These data clearly indicate the possibility that simultaneously inhibiting and/or activating different Y receptors with a combination of Y receptor antagonists and agonists could provide new treatments to improve the efficiency of lifestyle interventions for obesity.
ACKNOWLEDGMENTS
Supported in part by grant no. 188 827 from the National Health and Medical Research Council of Australia (NH&MRC) and the Diabetes Australia Research Trust (to A.S.) and grant no. 230 820 to A.S. from the NH&MRC.
The authors thank Professor T.G. Redgrave (Department of Physiology, University of Western Australia, Crawley, Australia) for advice on analysis of fecal lipids, Dr. Christine Biben (Victor Chang Cardiac Research Institute, Sydney, Australia) for help with analysis of intestinal morphology, Dr. Paul Baldock (Bone and Mineral Research Program, Garvan Institute, Darlinghurst, Australia) for discussion about the manuscript, and Dr. Julie Ferguson (Biological Testing Facility, Garvan Institute, Darlinghurst, Australia) for facilitation of mouse studies.
FOOTNOTES
H.T.B. is currently affiliated with the Department of Human Anatomy and Cell Science, University of Manitoba, Winnipeg, Canada.
A.S. and H.T.B. contributed equally to this work.
REFERENCES
Batterham RL, Cowley MA, Small CJ, Herzog H, Cohen MA, Dakin CL, Wren AM, Brynes AE, Low MJ, Ghatei MA, Cone RD, Bloom SR: Gut hormone peptide YY3eC36 physiologically inhibits food intake. Nature 418:650eC654, 2002
Iyengar S, Li DL, Simmons RM: Characterization of neuropeptide Y-induced feeding in mice: do Y1eCY6 receptor subtypes mediate feeding J Pharmacol Exp Ther 289:1031eC1040, 1999
Pedrazzini T, Seydoux J, Kunstner P, Aubert JF, Grouzmann E, Beermann F, Brunner HR: Cardiovascular response, feeding behavior and locomotor activity in mice lacking the neuropeptide Y Y1 receptor. Nat Med 4:722eC726, 1998
Pralong FP, Gonzales C, Voirol MJ, Palmiter RD, Brunner HR, Gaillard RC, Seydoux J, Pedrazzini T: The neuropeptide Y Y1 receptor regulates leptin-mediated control of energy homeostasis and reproductive functions. FASEB J 16:712eC714, 2002
Kushi A, Sasai H, Koizumi H, Takeda N, Yokoyama M, Nakamura M: Obesity and mild hyperinsulinemia found in neuropeptide Y-Y1 receptor-deficient mice. Proc Natl Acad Sci U S A 95:15659eC15664, 1998
Sainsbury A, Schwarzer C, Couzens M, Fitissov S, Furtinger S, Jenkins A, Cox HM, Sperk G, Hokfelt T, Herzog H: Important role of hypothalamic Y2 receptors in bodyweight regulation revealed in conditional knockout mice. Proc Natl Acad Sci U S A 99:8938eC8943, 2002
Sainsbury A, Schwarzer C, Couzens M, Herzog H: Y2 receptor deletion attenuates the type 2 diabetic syndrome of ob/ob mice. Diabetes 51:3420eC3427, 2002
Naveilhan P, Svensson L, Nystrom S, Ekstrand AJ, Ernfors P: Attenuation of hypercholesterolemia and hyperglycemia in ob/ob mice by neuropeptide Y Y2 receptor ablation. Peptides 23:1087eC1091, 2002
Naveilhan P, Hassani H, Canals JM, Ekstrand AJ, Larefalk A, Chhajlani V, Arenas E, Gedda K, Svensson L, Thoren P, Ernfors P: Normal feeding behavior, body weight and leptin response require the neuropeptide Y Y2 receptor. Nat Med 5:1188eC1193, 1999
Leibowitz SF, Alexander JT: Analysis of neuropeptide Y-induced feeding: dissociation of Y1 and Y2 receptor effects on natural meal patterns. Peptides 12:1251eC1260, 1991
Halatchev IG, Cone RD: Peripheral administration of peptide YY3eC36 produces conditioned taste aversion in mice. Cell Metab 1:159eC168, 2005
Batterham RL, Cohen MA, Ellis SM, Le Roux CW, Withers DJ, Frost GS, Ghatei MA, Bloom SR: Inhibition of food intake in obese subjects by peptide YY3eC36. N Engl J Med 349:941eC948, 2003
Asakawa A, Inui A, Yuzuriha H, Ueno N, Katsuura G, Fujimiya M, Fujino MA, Niijima A, Meguid MM, Kasuga M: Characterization of the effects of pancreatic polypeptide in the regulation of energy balance. Gastroenterology 124:1325eC1336, 2003
Gettys TW, Garcia R, Savage K, Whitcomb DC, Kanayama S, Taylor IL: Insulin-sparing effects of pancreatic polypeptide in congenitally obese rodents. Pancreas 6:46eC53, 1991
Ueno N, Inui A, Iwamoto M, Kaga T, Asakawa A, Okita M, Fujimiya M, Nakajima Y, Ohmoto Y, Ohnaka M, Nakaya Y, Miyazaki JI, Kasuga M: Decreased food intake and body weight in pancreatic polypeptide-overexpressing mice. Gastroenterology 117:1427eC1432, 1999
Sainsbury A, Schwarzer C, Couzens M, Jenkins A, Oakes SR, Ormandy CJ, Herzog H: Y4 receptor knockout rescues fertility in ob/ob mice. Genes Dev 16:1077eC1088, 2002
Lin S, Boey D, Couzens M, Lee N, Sainsbury A, Herzog H: Compensatory changes in [125I]-peptide YY binding in Y receptor knockout mice suggest the potential existence of further Y receptor(s). Neuropeptides 39:21eC28, 2005
Howell OW, Scharfman HE, Beck-Sickinger AG, Herzog H, Gray WP: Neuropeptide Y is neuroproliferative in primary hippocampal cultures. J Neurochemistry 86:646eC659, 2003
Tschenett A, Singewald N, Carli M, Balducci C, Salchner P, Vezzani A, Herzog H, Sperk G: Reduced anxiety and improved stress coping ability in mice lacking neuropeptide Y-Y2 receptors. Euro J Neurosci 18:143eC148, 2003
Baran K, Preston E, Wilks D, Cooney GJ, Kraegen EW, Sainsbury A: Chronic central melanocortin-4 receptor antagonism and central neuropeptide-y infusion in rats produce increased adiposity by divergent pathways. Diabetes 51:152eC158, 2002
Yu C, Wang F, Kan M, Jin C, Jones RB, Weinstein M, Deng CX, McKeehan WL: Elevated cholesterol metabolism and bile acid synthesis in mice lacking membrane tyrosine kinase receptor FGFR4. J Biol Chem 275:15482eC15489, 2000
Folch JMS, Less M, Stanley GHS: A simple method for the isolation and purification of total lipids from animal tissue. J Biol Chem 226:497eC509, 1956
Carlson LA: Determination of serum triglycerides. J Atheroscler Res 37:334eC336, 1963
Ricquier D, Bouillaud F, Toumelin P, Mory G, Bazin R, Arch J, Penicaud L: Expression of uncoupling protein mRNA in thermogenic or weakly thermogenic brown adipose tissue: evidence for a rapid beta-adrenoreceptor-mediated and transcriptionally regulated step during activation of thermogenesis. J Biol Chem 261:13905eC13910, 1986
Sainsbury A, Baldock PA, Schwarzer C, Ueno N, Enriquez RF, Couzens M, Inui A, Herzog H, Gardiner EM: Synergistic effects of Y2 and Y4 receptors on adiposity and bone mass revealed in double knockout mice. Mol Cell Biol 23:5225eC5233, 2003
Colombo C, Arlati S, Curcio L, Maiavacca R, Garatti M, Ronchi M, Corbetta C, Giunta A: Effect of taurine supplementation on fat and bile acid absorption in patients with cystic fibrosis. Scand J Gastroenterol Suppl 143:151eC156, 1988
Furuse M, Mabayo RT, Okumura J: Effects of dietary cholestyramine on the utilisation of diets containing medium or long chain triacylglycerols by chicks. Br Poult Sci 38:436eC438, 1997
Hoentjen F, Hopman WP, Jansen JB: Effect of circulating peptide YY on gallbladder emptying in humans. Scand J Gastroenterol 36:1086eC1091, 2001
Adrian TE, Mitchenere P, Sagor G, Bloom SR: Effect of pancreatic polypeptide on gallbladder pressure and hepatic bile secretion. Am J Physiol 243:G204eCG207, 1982
Pomeranz IS, Davison JS, Shaffer EA: In vitro effects of pancreatic polypeptide and motilin on contractility of human gallbladder. Dig Dis Sci 28:539eC544, 1983
Satchithanandam S, Vargofcak-Apker M, Calvert RJ, Leeds AR, Cassidy MM: Alteration of gastrointestinal mucin by fiber feeding in rats. J Nutr 120:1179eC1184, 1990
Pettersson M, Ahreen B, Lundquist I, Bttcher G, Sundler F: Neuropeptide Y: intrapancreatic neuronal localization and effects on insulin secretion in the mouse. Cell Tissue Res 248:43eC48, 1987
Moltz JH, McDonald JK: Neuropeptide Y: direct and indirect action on insulin secretion in the rat. Peptides 6:1155eC1159, 1985
Morgan DG, Kulkarni RN, Hurley JD, Wang ZL, Wang RM, Ghatei MA, Karlsen AE, Bloom SR, Smith DM: Inhibition of glucose stimulated insulin secretion by neuropeptide Y is mediated via the Y1 receptor and inhibition of adenylyl cyclase in RIN 5AH rat insulinoma cells. Diabetologia 41:1482eC1491, 1998
Cho YR, Kim CW: Neuropeptide Y promotes beta-cell replication via extracellular signal-regulated kinase activation. Biochem Biophys Res Commun 314:773eC780, 2004
Standridge M, Alemzadeh R, Zemel M, Koontz J, Moustaid-Moussa N: Diazoxide down-regulates leptin and lipid metabolizing enzymes in adipose tissue of Zucker rats. FASEB J 14:455eC460, 2000
Burcelin R, Brunner H, Seydoux J, Thorensa BTP: Increased insulin concentrations and glucose storage in neuropeptide Y Y1 receptor-deficient mice. Peptides 22:421eC427, 2001
Onaga T, Zabielski R, Kato S: Multiple regulation of peptide YY secretion in the digestive tract. Peptides 23:279eC290, 2002
Imamura M: Effects of surgical manipulation of the intestine on peptide YY and its physiology. Peptides 23:403eC407, 2002
Challis BG, Pinnock SB, Coll AP, Carter RN, Dickson SL, O’Rahilly S: Acute effects of peptide YY3eC36 on food intake and hypothalamic neuropeptide expression in the mouse. Biochem Biophys Res Commun 311:915eC919, 2003
Stephens TW, Basinski M, Bristow PK, Bue-Valleskey JM, Burgett SG, Craft L, Hale J, Hoffmann J, Hsiung HM, Kriauciunas A, MacKellar W, Rosteck PR, Schoner B, Smith D, Tinsley FC, Zhang XY, Heiman M: The role of neuropeptide Y in the antiobesity action of the obese gene product. Nature 377:530eC532, 1995
Guan XM, Yu H, Van der Ploeg LH: Evidence of altered hypothalamic pro-opiomelanocortin/ neuropeptide Y mRNA expression in tubby mice. Brain Res Mol Brain Res 59:273eC279, 1998
Mizuno TM, Kleopoulos SP, Bergen HT, Roberts JL, Priest CA, Mobbs CV: Hypothalamic pro-opiomelanocortin mRNA is reduced by fasting and [corrected] in ob/ob and db/db mice, but is stimulated by leptin. Diabetes 47:294eC297, 1998
Huang XF, Han M, South T, Storlien L: Altered levels of POMC, AgRP and MC4-R mRNA expression in the hypothalamus and other parts of the limbic system of mice prone or resistant to chronic high-energy diet-induced obesity. Brain Res 992:9eC19, 2003(Amanda Sainsbury, Hugo T.)
2 Diabetes Research Program, The Garvan Institute of Medical Research, St. Vincent’s Hospital, Sydney, Australia
3 Department of Pharmacology and Toxicology, University of Innsbruck, Innsbruck, Austria
4 Neuroscience Institute of Schizophrenia and Allied Disorders, Sydney, Australia
5 Bone and Mineral Research Program, The Garvan Institute of Medical Research, St. Vincent’s Hospital, Sydney, Australia
6 Department of Pharmacology, Innsbruck Medical University, Innsbruck, Austria
POMC, proopiomelanocortin; WATe, epididymal white adipose tissue
ABSTRACT
Neuropeptide Y receptors are critical regulators of energy homeostasis, but the functional interactions and relative contributions of Y receptors and the environment in this process are unknown. We measured the effects of an ad libitum diet of normal or high-fat food on energy balance in mice with single, double, or triple deficiencies of Y1, Y2, or Y4 receptors. Whereas wild-type mice developed diet-induced obesity, Y2Y4 double knockouts did not. In contrast, Y1 knockout or Y1Y2 or Y1Y4 receptor double knockout mice developed an exacerbated diet-induced obesity syndrome. Remarkably, the antiobesity effect of Y2Y4 deficiency was stronger than the obesogenic effect of Y1 deficiency, since Y1Y2Y4 triple knockouts did not develop obesity on the high-fat diet. Resistance to diet-induced obesity in Y2Y4 knockouts was associated with reduced food intake and improved glucose tolerance in the absence of changes in total physical activity. Fecal concentration of free fatty acids was significantly increased in Y2Y4 knockouts in association with a significantly reduced bile acid pool and marked alterations in intestinal morphology. In addition, hypothalamic proopiomelanocortin expression was decreased in diet-induced obesity (in both wild-type and Y1 receptor knockout mice) but not in obesity-resistant Y2Y4 receptor knockout mice fed a high-fat diet. Therefore, deletion of Y2 and Y4 receptors synergistically protects against diet-induced obesity, at least partially via changes in food intake and hypothalamic proopiomelanocortin expression.
Members of the Y receptor family, notably Y1, Y2, Y4, and Y5 receptors, are implicated in energy homeostasis and the development of obesity and insulin resistance. These receptors are activated by three endogenous ligands: neuropeptide Y, the gut-derived hormones peptide YY, and pancreatic polypeptide. Recently, there has been renewed speculation that ligands for Y receptors, such as peptide YY3eC36 and pancreatic polypeptide, may be of benefit for the treatment of obesity (1).
There is considerable conflict in the literature about the role of Y receptors in the regulation of body weight. For instance, pharmacological studies suggested that Y1 receptors contribute to hyperphagia induced by increased hypothalamic neuropeptide Y secretion (2). Fasting-induced refeeding is reduced in Y1 knockout mice (3). Moreover, food intake and body weight of genetically obese ob/ob mice, in which hypothalamic neuropeptide Y-ergic activity is chronically increased, are significantly reduced by Y1 knockout (4). In contrast, Y1 knockouts develop significant increases in body weight, fat mass, and insulinemia in the absence of hyperphagia (3,5).
Similar controversies prevail regarding the role of Y2 receptors in energy homeostasis. Hypothalamus-specific or germ-line deletion of Y2 receptors resulted in significant reductions in the body weight of lean mice (6) and significant reductions in adiposity or body weight and the type 2 diabetic syndrome of ob/ob mice in the absence of reductions in food intake (7,8). In contrast, another germ-line Y2 receptor knockout model was shown to develop increased body weight, fat deposition, and hyperphagia (9). Furthermore, intrahypothalamic administration of the Y2 agonist neuropeptide Y[13eC36] to rats significantly decreased food intake (10). Moreover, circulating peptide YY from the gut and its truncated form peptide YY3eC36 (a preferential Y2 agonist) have been shown to reduce food intake, adiposity, and/or body weight after short- or long-term administration to lean or obese rodents or humans (1,11,12).
Recent work suggests that pancreatic polypeptide, a preferential Y4 receptor agonist secreted by the F cells of the islets of Langerhans, regulates energy homeostasis. Intravenous or intraperitoneal administration of pancreatic polypeptide to mice increases metabolic rate (13) and decreases hyperglycemia, basal or glucose-induced hyperinsulinemia, insulin resistance, and hyperlipidemia in ob/ob mice, culminating in decreases in adiposity and body weight (13,14). Pancreatic polypeptide transgenic mice, which have a 20-fold increase in plasma pancreatic polypeptide levels, have reduced food intake, body weight, and fat mass as well as reduced glucose-induced insulin secretion and gastric emptying when compared with control mice (15). However, other findings do not support a role of Y4 receptors in energy homeostasis. Y4 receptor knockout had no effect on the hyperphagia, obesity, or type 2 diabetic phenotype of ob/ob mice (16).
These conflicting data make it difficult to define the true physiological and pathophysiological role of Y receptors in energy homeostasis and obesity. Moreover, it is difficult to know whether potential obesity treatments targeted to Y receptors should act as agonists or antagonists. Part of this conflict can be explained by the fact that Y receptors mediate their physiological effects via complex synergistic and redundant interactions. For instance, knockout of individual Y receptors alters ligand-binding patterns of the remaining Y receptors (17). In addition, ligands for Y receptors are renowned for their lack of specificity for individual receptor types.
To circumvent some of these complications of Y receptor biology, we made a systematic study of energy balance in mice with single, double, or triple knockout of Y1, Y2, or Y4 receptors, both under chow-fed conditions and in diet-induced obesity. In this way, we were able to clarify the interactions among these receptor types in regulating energy balance (including body weight, food intake, physical activity, thermogenesis, glucose tolerance, intestinal lipid absorption, intestinal morphology, and hypothalamic expression of neuropeptide Y and proopiomelanocortin) and to elucidate their roles in the development of diet-induced obesity. Y5 receptor knockouts were not investigated in this study because the Y5 and Y1 receptors are localized only 20 kb apart on the same chromosome, and it is therefore not plausible to obtain Y1Y5 receptor double knockout mice by cross-breeding.
RESEARCH DESIGN AND METHODS
Generation of the Y1, Y2, and Y4 knockout mice were previously published (6,16,18). Male double or triple knockout mice were obtained by crossing Y1, Y2, or Y4 knockout mice, respectively. All mice were on a mixed C57BL/6-129/SvJ background. All research and animal care procedures were approved by the Garvan Institute/St. Vincent’s Hospital Animal Experimentation Ethics Committee and were in agreement with the Australian Code of Practice for the Care and Use of Animals for Scientific Purpose. Mice were housed under conditions of controlled temperature (22°C) and illumination (12-h light cycle, lights on at 0700). Half of the mice of each genotype were fed a normal chow diet ad libitum (6% calories from fat, 21% calories from protein, 71% calories from carbohydrate, and 2.6 kcal/g; Gordon’s Speciality Stock Feeds, Yanderra, NSW, Australia). The other half was fed ad libitum with a high-fat diet supplemented with fat and sucrose (46% calories from fat, 21% calories from protein, 33% calories from carbohydrate, and 4.72 kcal/g) from 8 weeks of age onwards. The diet was based on the composition of rodent diet catalog no. D12451 (Research Diets, New Brunswick, NJ), with the exception that safflower oil and copha were used in place of soybean oil and lard. Twenty-four-hour food intake was estimated twice a week for 4 weeks from 13 to 16 weeks of age by the average amount of food removed from the food hopper. In a subset of animals, the actual proportion of food that was consumed was calculated by subtracting the weight of the food spilt in the cage from the weight of food removed from the hopper. Body temperature was determined at 14eC16 weeks of age (BAT-10 multipurpose thermometer; Physitemp Instruments, Clifton, NJ).
Determination of physical activity.
Distance traveled in the open field test was determined as previously described (19). Total cage activity was recorded using a passive infrared detector (Conrad Electronics, Hanover, Germany) on top of the cage lid of single-housed animals. The signals of the passive infrared detectors were detected by an I/O interface card (PIO48 II; Conrad Electronics) and stored using custom-made software. A mean activity per day was calculated by averaging corresponding 30-min intervals of 7 consecutive days.
Glucose tolerance tests and tissue collection.
At 16 weeks of age, animals were fasted overnight and then administered an intraperitoneal injection of D-glucose (60 mg/mouse). Glucose levels were determined in tail blood samples with glucose test strips (Accu-Check Advantage II Glucose Test Strips; Roche Diagnostics Australia, Castle Hill, NSW, Australia). At 3eC8 days after the glucose tolerance test, food was removed from cage hoppers at 0900, and animals were killed 4eC7 h later by cervical dislocation followed by cardiac puncture for serum collection. Brains were immediately removed and frozen on dry ice. The interscapular brown adipose tissue was removed, weighed, and stored frozen until analysis for uncoupling protein-1 mRNA levels (20). Epididymal white adipose tissue (WATe) depots were dissected and weighed. The small intestine of some mice was removed and prepared for histology as described below. In separate groups of mice, the small intestine was flushed with isotonic saline, blotted on paper towels, and weighed. In other mice, the liver, gallbladder, small intestine and contents were removed, weighed, and stored at eC80°C until analysis of total bile acid pool (21).
Analysis of fecal lipids and bile acids.
Fecal samples were dried to a constant weight at 90°C. Some fecal samples were used for extraction of total lipids (22). Lipids were resuspended in pure chloroform and separated by TLC (thin-layer chromatography) on Whatman Silica Gel K6 glass TLC plates (Whatman, Maidstone, England) in hexane:diethyl ether:acetic acid, 80:20:1. Standards were triolein (50 e蘥), dipalmitine (75 e蘥), and palmitine (125 e蘥) (Sigma, St. Louis, MO). Fractions corresponding to triglycerides, diglycerides, monoglycerides, and free fatty acids were scraped from the plate for assay of triacylglycerol (23). The free fatty acid fraction was resuspended in chloroform and assayed for free fatty acids with a colorimetric assay kit (WAKO, Saitama, Japan). Other samples of feces were used for determination of fecal bile acid concentration (21).
Analysis of intestinal morphology.
One-centimeter pieces of small intestine were taken from the following three locations: duodenum, 3 cm distal from the stomach-duodenal junction; jejunum, at the midpoint of the small intestine; and ileum, 3 cm proximal from the ileocaecal junction. Intestinal pieces were fixed for 16eC18 h in Bouin’s solution (Sigma), embedded in paraffin, sectioned at 7 e蘭, mounted on charged slides (SuperFrost Plus; Menzel-Glaser, Braunschweig, Germany), and stained with periodic acid Schiff. Villus length (from base of the crypt to tip of the villus) was measured in at least 15 villi from three mice in each group, using a light microscope (Axiophot; Zeiss, Oberkochen, Germany) connected to a Leica camera and Leica Image Manager software (Leica, Heerbrugg, Switzerland). The number of goblet cells per villus (excluding crypts) were counted in at least 15 villi from five mice in each group. All analyses were done by investigators unaware of genotype.
Neurochemical analyses.
Coronal brain slices (20 e蘭) at the level of the hypothalamic arcuate nucleus were hybridized using DNA oligonucleotides complementary to mouse neuropeptide Y and proopiomelanocortin (POMC) (7).
Serum analyses.
Serum hormone levels were determined with commercial radioimmunoassay kits from Linco Research (St. Louis, MO) (leptin and insulin), ICN Biomedicals (Costa Mesa, CA) (corticosterone, free T4, and testosterone), and Bioclone Australia (Marrickville, Australia) (IGF-1). Basal serum glucose levels were determined with a glucose oxidase kit (Trace Scientific, Melbourne, Australia).
Statistical analysis.
All data are expressed as means ± SE. Differences among groups of mice were assessed by ANOVA or repeated-measures ANOVA, followed by Fisher’s post hoc comparisons, if appropriate (StatView version 4.51; Abacus Concepts, Berkeley, CA). Statistical significance was defined as P < 0.05.
RESULTS
Effects of Y receptor deficiency on diet-induced obesity.
The body weights of mice at 8 weeks of age (before the introduction of the high-fat diet) were not significantly different from each other, except for Y1Y2Y4 triple knockout mice, which were lighter (wild-type, 23.7 ± 0.3 g; Y1, 22.8 ± 0.6 g; Y1Y2, 24.0 ± 0.4 g; Y1Y4, 24.2 ± 0.4 g; Y2, 24.5 ± 0.6 g; Y4, 24.4 ± 0.3 g; Y2Y4, 24.8 ± 0.4 g; and Y1Y2Y4, 22.1 ± 0.5 g, P < 0.05 vs. wild-type, n = at least 11 mice per genotype). Figure 1A, shows that under chow-fed conditions, body weight gain from 8 weeks until 16 weeks of age was similar in all groups of mice, except for Y1 knockout and Y1Y4 double knockout mice. The absolute and relative weights of the WATe depot and serum leptin levels were similar in all groups of mice under chow-fed conditions, except for Y2Y4 double and Y1Y2Y4 triple knockout mice, which showed significant decreases (Fig. 1B and C, Fig. 2A, ). Under chow-fed conditions, serum insulin levels were significantly increased over wild-type values in Y1 and Y1Y4 double knockout mice, and significantly decreased relative to wild-type in Y2Y4 double knockouts (Fig. 2B, ). In chow-fed animals, there were no significant differences among groups with respect to serum levels of corticosterone or free T4 (Fig. 1C and D), glucose, testosterone, or IGF-1 (data not shown). When fed ad libitum for 8 weeks on the high-fat diet, wild-type mice developed a distinct obesity syndrome characterized by increased body weight gain (Fig. 1A), increased WATe weight (Fig. 1B and C), and increases in serum leptin, insulin, and corticosterone levels (Fig. 2AeCC), with a tendency to decreased free T4 levels (Fig. 2D). Y1 receptor deficiency, either alone or in combination with Y2 or Y4 receptor deficiency (i.e., Y1, Y1Y2, and Y1Y4 receptor knockout mice), exacerbated this diet-induced obesity syndrome (Figs. 1AeCC and 2B). Y1, Y1Y2, and Y1Y4 receptor knockout mice also developed hyperleptinemia, hypercorticosteronemia, and a tendency to decreased free T4 levels after high-fat feeding, but these responses were not significantly different from wild-type animals (Fig. 2A,C,D). In contrast to the exacerbating effect of Y1 deficiency on diet-induced obesity, Y2 or Y4 deletion (in Y2, Y4, Y2Y4 double, and Y1Y2Y4 triple knockout mice) conferred protection against diet-induced obesity (Figs. 1 and 2). However, similar to wild-type mice, Y2, Y2Y4 double, and Y1Y2Y4 triple knockout mice showed marked decreases in serum free T4 levels in response to high-fat feeding (Fig. 2D).
Effects of Y receptor deficiency on feeding behavior and food intake.
As shown by the open columns in Fig. 3, Y1Y4, Y4, Y2Y4 double, and Y1Y2Y4 triple knockout mice removed significantly more normal chow from the hopper everyday compared with wild-type mice. When fed the high-fat diet, Y1Y4, Y2Y4 double, and Y1Y2Y4 triple knockout mice removed significantly more high-fat diet from the cage hopper than wild-type controls (Fig. 3, ). Y2Y4 double knockout mice spilled significantly more of the food taken from the cage hopper than wild-type mice, and the amount of chow or high-fat food actually consumed was significantly less than that consumed by wild-types (Fig. 4). There was no significant effect of diet or genotype on water intake (data not shown). Because double deletion of Y2 and Y4 receptors conferred resistance against diet-induced obesity (even in combination with Y1 receptor deficiency), we focused our attention from here onwards on the Y2Y4 knockouts.
No effect of Y2Y4 receptor double knockout on physical activity or thermogenesis.
In the open field test, there was no significant difference between Y2Y4 double knockout mice and wild-type controls with respect to total distance traveled (6,960 ± 510 vs. 5,830 ± 240 cm/30 min, respectively; n = at least 4 mice per group). Moreover, there was no significant difference between double knockout and wild-type mice with respect to total physical activity in the home cage (data not shown).
As an index of nonactivity thermogenic capacity, we measured expression of uncoupling protein-1 mRNA in brown adipose tissue (24) and rectal temperature in Y2Y4 double knockout and wild-type mice. There was no significant difference between groups with respect to uncoupling protein-1 mRNA levels in brown adipose tissue, standardized to glyceraldehyde-3-phosphate dehydrogenase mRNA levels and expressed as a percent of wild-type values (107.3 ± 6.7 vs. 100.0 ± 7.4% in Y2Y4 double knockout and wild-type mice, respectively; n = 5 mice per group). Moreover, rectal temperature of chow- or fat-fed Y2Y4 receptor double knockout mice was not significantly different from that of wild-type mice fed on the same diet (data not shown).
Enhanced glucose tolerance in Y2Y4 receptor double knockout mice.
There was no significant difference among groups with respect to peak glycemia following intraperitoneal glucose injection. However, in Y2, Y2Y4 double, and Y1Y2Y4 triple knockout mice, there was a significant reduction in serum glucose levels at 60 and 90 min postinjection compared with wild-type mice (Fig. 5). All other groups of mice (Y1, Y1Y2 double, Y1Y4 double, and Y4 knockout mice) showed glucose clearance curves indistinguishable from wild-type mice (Fig. 5).
Decreased intestinal lipid absorption in Y2Y4 receptor double knockout mice.
Fig. 6 shows the concentration of triglycerides, diglycerides, and free fatty acids in feces from wild-type and Y2Y4 receptor double knockout mice, either under chow-fed (Fig. 6A) or high-fateCfed (Fig. 6B) conditions. In both groups of mice, free fatty acids were the most abundant fecal lipid species, and undigested or partially digested lipids (triglycerides and diglycerides) were 6- to 30-fold less abundant. Monoglycerides were not detectable in the fecal samples investigated. These findings indicate that triglyceride digestion to free fatty acids proceeds efficiently in both wild-type and Y2Y4 receptor double knockout mice. However, absorption of digested lipids may be impaired in Y2Y4 double knockout mice, since the fecal concentration of free fatty acids was increased in knockout versus wild-type mice, significantly so during high-fat feeding (Fig. 6B). Daily fecal output (dried weight) from Y2Y4 double knockout mice on a chow diet was significantly less than that of wild-type controls (0.64 ± 0.12 vs. 1.23 ± 0.04 g · mouseeC1 · dayeC1, n = 3eC4 cages of 2eC4 mice per group). However, there was no significant difference in daily fecal output between knockout and wild-type mice on the high-fat diet (0.22 ± 0.02 vs. 0.25 ± 0.04 g · mouseeC1 · dayeC1 in wild-types, n = 3eC4 cages of 2eC4 mice per group).
Reduced bile pool and differences in intestinal morphology in Y2Y4 receptor double knockout mice.
We determined the total bile pool because intestinal lipid absorption is dependent on bile production. The total bile pool of Y2Y4 knockouts was significantly reduced compared with wild-type mice (20.0 ± 3.6 vs. 41.0 ± 3.0 e蘭ol total bile, respectively; and 88 ± 17 vs. 142 ± 10 e蘭ol/100 g body wt, respectively; means ± SE of 12eC13 mice per group, P < 0.01). Fecal total bile acid concentration was similar in Y2Y4 knockout and wild-type mice (33 ± 9 vs. 34 ± 2 e蘭ol/g dried feces, respectively, means ± SE of at least three mice per group). Since daily fecal output was reduced by 50% in Y2Y4 versus wild-type mice, total bile acid output of Y2Y4 knockouts was therefore half that of wild-type counterparts, in keeping with the smaller total bile acid pool size.
Although there was no significant difference between wild-type and knockout mice with regards to either weight (1.1 ± 0.05 vs. 1.1 ± 0.05 g, respectively; means ± SE of 22eC24 mice per group) or length (39.4 ± 0.8 vs. 37.6 ± 0.6 cm, respectively; means ± SE of 25eC27 mice per group) of the small intestine, the average length of small intestinal villi was significantly decreased in the duodenum of Y2Y4 double knockout versus wild-type mice (431 ± 2 vs. 525 ± 6 e蘭, respectively, n = 3 mice per genotype, P < 0.05) (Fig. 7A and B). This difference in villus length was not observed in the jejunum (295 ± 16 vs. 287 ± 6 e蘭; Y2Y4 and wild-type mice, respectively, n = 3 mice per genotype) or ileum (204 ± 24 vs. 184 ± 15 e蘭 in Y2Y4 and wild-type mice, respectively, n = 3 mice per genotype). Y2Y4 receptor double knockout mice had an increase over wild-type mice in the number of goblet cells in the duodenum (23.7 ± 1.7 vs. 20.6 ± 1.0 goblet cells per villus in wild-type mice, n = 5 mice per group, NS), the jejunum (21.6 ± 1.1 vs. 18.0 ± 0.6 goblet cells per villus in wild-type mice, n = 5 mice per group, P < 0.05), and the ileum (23.1 ± 1.4 vs. 18.2 ± 0.6 goblet cells per villus in wild-type mice, n = 5 mice per group, P < 0.05). Representative villi from the ileum of wild-type and Y2Y4 receptor knockout mice are shown in Fig. 7C and D.
Altered hypothalamic expression of neuropeptide Y and POMC in Y receptor knockout mice.
In wild-type mice, the high-fat diet resulted in significant decreases in neuropeptide Y and POMC mRNA levels in the arcuate nucleus (Table 1). Increased weight gain in both chow-fed and high-fateCfed Y1 receptor knockout mice, compared with wild-types, was associated with decreased neuropeptide Y and POMC mRNA levels compared with wild-type values (Table 1). In addition, in Y1 receptor knockout mice, diet-induced obesity was associated with decreased neuropeptide Y and POMC mRNA levels compared with chow-fed mice (Table 1). In contrast, in Y2Y4 receptor knockout mice, neuropeptide Y and POMC mRNA levels were not significantly decreased following ingestion of a high-fat diet compared with chow-fed double knockouts. Rather, neuropeptide Y and POMC mRNA levels were increased in Y2Y4 receptor knockout mice compared with wild-type mice fed the same diet (Table 1).
DISCUSSION
These data demonstrate that ablation of Y2 and Y4 receptors protects against diet-induced obesity, probably via reduced food intake. In contrast, deletion of Y1 receptors significantly exacerbates diet-induced obesity. Moreover, the antiobesity effect of double Y2 and Y4 receptor deficiency overrides the obesogenic effect of Y1 receptor deficiency and a high-fat diet (as seen in Y1Y2Y4 triple knockout mice). These observations provide evidence that the molecular system that regulates energy homeostasis, including the Y receptor system, has less redundancy and capacity for compensation to favor positive over negative energy balance than previously thought.
We recently showed that Y2 and Y4 receptor knockout synergistically increases bone volume in conjunction with a significant increase in bone formation rate (25). It is therefore conceivable that in addition to decreased food intake, this threefold increase in bone volume could contribute to the lean phenotype observed in Y2Y4 receptor double knockout mice.
The current work suggests that Y2Y4 receptor double deficiency decreases absorption of fat from the digestive tract. This change was not associated with any decrease in efficiency of triglyceride digestion to free fatty acids. The reduced intestinal fat absorption in Y2Y4 receptor knockout mice is likely to be mediated, at least in part, by the reduced total bile pool observed in these animals. Indeed, fat absorption and energy extraction from food is positively correlated with the concentration of functional bile acids (26,27). It is unlikely however that decreased intestinal lipid absorption contributes to the lean phenotype of Y2Y4 receptor knockout mice, since the calorie content of the extra free fatty acids excreted in the feces of these mice represents <1% their total daily calorie intake. Previous work has shown that both peptide YY and pancreatic polypeptide inhibit gallbladder emptying (28) or gallbladder bile output in pigs and/or humans (28,29), presumably via interactions with Y receptors in the dorsovagal complex of the brain stem and subsequent vagal-dependent pathways (28,30). The current work extends these data to show the unambiguous involvement of Y2 and Y4 receptors in the regulation of total bile pool.
Reduced intestinal absorption of lipids in Y2Y4 receptor double knockout mice may be influenced by the observed decrease in villus length of the duodenum. It is also possible that the increased number of goblet cells observed in the small intestine of Y2Y4 receptor double knockout mice may have led to increased production of intestinal mucus, which could accelerate intestinal transit and impair absorption of nutrients (31).
In contrast to the protective effects of Y2 and Y4 receptor deficiency against diet-induced obesity, animals deficient in Y1 receptors had a tendency for increased adiposity under chow-fed conditions and had an exacerbated obesity syndrome in response to the high-fat diet. This was observed in single Y1 receptor knockout animals as well as in Y1Y2 and Y1Y4 double knockout animals. In Y1Y2Y4 receptor triple knockout animals however, the antiobesity effect of Y2 and Y4 receptor deficiency predominated over the obesogenic effect of Y1 deficiency and a high-fat diet. It is likely that hormonal and metabolic alterations, notably hyperinsulinemia, contribute to the increased propensity for obesity in Y1, Y1Y2, and Y1Y4 receptor knockout mice. When injected peripherally or when administered to pancreatic tissue in vitro, neuropeptide Y decreases insulin secretion (32,33), probably via direct interaction with the Y1 receptors expressed on pancreatic -cells (34,35). It is likely that the lack of Y1 receptors on pancreatic islet tissue contributed to the increased basal serum insulin levels of Y1, Y1Y2, and Y1Y4 receptor knockout mice, with hyperinsulinemia subsequently contributing to increased adiposity (36). Indeed, we have shown that glucose-induced serum insulin levels are increased in Y1 knockout compared with wild-type mice (A.S., N.J.L., K.S., R.E., and H.H., unpublished data). Other groups have also reported that germline Y1 receptor knockout induces obesity and basal hyperinsulinemia (3,5,37) and that hyperinsulinemia is likely to be a key etiological feature in the obese phenotype of these mice (37).
High-fat feeding resulted in decreased expression of neuropeptide Y in the arcuate nucleus of wild-type mice and Y1 receptor knockout mice, possibly as an adaptive response to inhibit further weight gain. This is most likely mediated via increased circulating concentrations of leptin and the gut peptide peptide YY3eC36, release of which is strongly increased by dietary fat (38,39), subsequently acting on leptin or Y2 receptors in the arcuate nucleus to reduce neuropeptide Y expression levels and thereby reducing feeding (1,40,41). Expression of POMC in the arcuate nucleus of wild-type and Y1 receptor knockout mice fed a high-fat diet was also reduced, a change that may contribute to the diet-induced obesity observed in these mice by decreasing secretion of -melanocyte stimulating hormone. Consistent with this hypothesis, rodent obesity models of genetic (42,43) or dietary (44) origin exhibit decreased expression of POMC in the arcuate nucleus. It was of interest that the decreases in hypothalamic expression of neuropeptide Y and POMC induced by the high-fat diet were absent in obesity-resistant Y2Y4 receptor knockout mice and were exacerbated in obesity-prone Y1 receptor knockout mice. These findings suggest a possible causative role of changes in POMC signaling in the development or resistance to diet-induced obesity in wild-type, Y1 knockout, and Y2Y4 receptor double knockout mice.
We recently demonstrated that single, double, or triple deletion of Y1, Y2, or Y4 receptors alters the expression or binding patterns of the remaining Y receptors in the brain (17). However, the major changes observed are located in areas not likely to participate in the regulation of energy homeostasis, notably the hippocampus. Therefore, it is unlikely that the changes in energy homeostasis observed in our panel of Y receptor knockout mice were due to compensatory changes in other Y receptors remaining in the hypothalamus.
In conclusion, these data demonstrate that Y2 and Y4 receptors are necessary for diet-induced obesity, at least in part via effects on food intake and hypothalamic POMC expression. In contrast, Y1 receptors inhibit diet-induced obesity, probably via direct inhibition of insulin secretion from pancreatic -cells. These data clearly indicate the possibility that simultaneously inhibiting and/or activating different Y receptors with a combination of Y receptor antagonists and agonists could provide new treatments to improve the efficiency of lifestyle interventions for obesity.
ACKNOWLEDGMENTS
Supported in part by grant no. 188 827 from the National Health and Medical Research Council of Australia (NH&MRC) and the Diabetes Australia Research Trust (to A.S.) and grant no. 230 820 to A.S. from the NH&MRC.
The authors thank Professor T.G. Redgrave (Department of Physiology, University of Western Australia, Crawley, Australia) for advice on analysis of fecal lipids, Dr. Christine Biben (Victor Chang Cardiac Research Institute, Sydney, Australia) for help with analysis of intestinal morphology, Dr. Paul Baldock (Bone and Mineral Research Program, Garvan Institute, Darlinghurst, Australia) for discussion about the manuscript, and Dr. Julie Ferguson (Biological Testing Facility, Garvan Institute, Darlinghurst, Australia) for facilitation of mouse studies.
FOOTNOTES
H.T.B. is currently affiliated with the Department of Human Anatomy and Cell Science, University of Manitoba, Winnipeg, Canada.
A.S. and H.T.B. contributed equally to this work.
REFERENCES
Batterham RL, Cowley MA, Small CJ, Herzog H, Cohen MA, Dakin CL, Wren AM, Brynes AE, Low MJ, Ghatei MA, Cone RD, Bloom SR: Gut hormone peptide YY3eC36 physiologically inhibits food intake. Nature 418:650eC654, 2002
Iyengar S, Li DL, Simmons RM: Characterization of neuropeptide Y-induced feeding in mice: do Y1eCY6 receptor subtypes mediate feeding J Pharmacol Exp Ther 289:1031eC1040, 1999
Pedrazzini T, Seydoux J, Kunstner P, Aubert JF, Grouzmann E, Beermann F, Brunner HR: Cardiovascular response, feeding behavior and locomotor activity in mice lacking the neuropeptide Y Y1 receptor. Nat Med 4:722eC726, 1998
Pralong FP, Gonzales C, Voirol MJ, Palmiter RD, Brunner HR, Gaillard RC, Seydoux J, Pedrazzini T: The neuropeptide Y Y1 receptor regulates leptin-mediated control of energy homeostasis and reproductive functions. FASEB J 16:712eC714, 2002
Kushi A, Sasai H, Koizumi H, Takeda N, Yokoyama M, Nakamura M: Obesity and mild hyperinsulinemia found in neuropeptide Y-Y1 receptor-deficient mice. Proc Natl Acad Sci U S A 95:15659eC15664, 1998
Sainsbury A, Schwarzer C, Couzens M, Fitissov S, Furtinger S, Jenkins A, Cox HM, Sperk G, Hokfelt T, Herzog H: Important role of hypothalamic Y2 receptors in bodyweight regulation revealed in conditional knockout mice. Proc Natl Acad Sci U S A 99:8938eC8943, 2002
Sainsbury A, Schwarzer C, Couzens M, Herzog H: Y2 receptor deletion attenuates the type 2 diabetic syndrome of ob/ob mice. Diabetes 51:3420eC3427, 2002
Naveilhan P, Svensson L, Nystrom S, Ekstrand AJ, Ernfors P: Attenuation of hypercholesterolemia and hyperglycemia in ob/ob mice by neuropeptide Y Y2 receptor ablation. Peptides 23:1087eC1091, 2002
Naveilhan P, Hassani H, Canals JM, Ekstrand AJ, Larefalk A, Chhajlani V, Arenas E, Gedda K, Svensson L, Thoren P, Ernfors P: Normal feeding behavior, body weight and leptin response require the neuropeptide Y Y2 receptor. Nat Med 5:1188eC1193, 1999
Leibowitz SF, Alexander JT: Analysis of neuropeptide Y-induced feeding: dissociation of Y1 and Y2 receptor effects on natural meal patterns. Peptides 12:1251eC1260, 1991
Halatchev IG, Cone RD: Peripheral administration of peptide YY3eC36 produces conditioned taste aversion in mice. Cell Metab 1:159eC168, 2005
Batterham RL, Cohen MA, Ellis SM, Le Roux CW, Withers DJ, Frost GS, Ghatei MA, Bloom SR: Inhibition of food intake in obese subjects by peptide YY3eC36. N Engl J Med 349:941eC948, 2003
Asakawa A, Inui A, Yuzuriha H, Ueno N, Katsuura G, Fujimiya M, Fujino MA, Niijima A, Meguid MM, Kasuga M: Characterization of the effects of pancreatic polypeptide in the regulation of energy balance. Gastroenterology 124:1325eC1336, 2003
Gettys TW, Garcia R, Savage K, Whitcomb DC, Kanayama S, Taylor IL: Insulin-sparing effects of pancreatic polypeptide in congenitally obese rodents. Pancreas 6:46eC53, 1991
Ueno N, Inui A, Iwamoto M, Kaga T, Asakawa A, Okita M, Fujimiya M, Nakajima Y, Ohmoto Y, Ohnaka M, Nakaya Y, Miyazaki JI, Kasuga M: Decreased food intake and body weight in pancreatic polypeptide-overexpressing mice. Gastroenterology 117:1427eC1432, 1999
Sainsbury A, Schwarzer C, Couzens M, Jenkins A, Oakes SR, Ormandy CJ, Herzog H: Y4 receptor knockout rescues fertility in ob/ob mice. Genes Dev 16:1077eC1088, 2002
Lin S, Boey D, Couzens M, Lee N, Sainsbury A, Herzog H: Compensatory changes in [125I]-peptide YY binding in Y receptor knockout mice suggest the potential existence of further Y receptor(s). Neuropeptides 39:21eC28, 2005
Howell OW, Scharfman HE, Beck-Sickinger AG, Herzog H, Gray WP: Neuropeptide Y is neuroproliferative in primary hippocampal cultures. J Neurochemistry 86:646eC659, 2003
Tschenett A, Singewald N, Carli M, Balducci C, Salchner P, Vezzani A, Herzog H, Sperk G: Reduced anxiety and improved stress coping ability in mice lacking neuropeptide Y-Y2 receptors. Euro J Neurosci 18:143eC148, 2003
Baran K, Preston E, Wilks D, Cooney GJ, Kraegen EW, Sainsbury A: Chronic central melanocortin-4 receptor antagonism and central neuropeptide-y infusion in rats produce increased adiposity by divergent pathways. Diabetes 51:152eC158, 2002
Yu C, Wang F, Kan M, Jin C, Jones RB, Weinstein M, Deng CX, McKeehan WL: Elevated cholesterol metabolism and bile acid synthesis in mice lacking membrane tyrosine kinase receptor FGFR4. J Biol Chem 275:15482eC15489, 2000
Folch JMS, Less M, Stanley GHS: A simple method for the isolation and purification of total lipids from animal tissue. J Biol Chem 226:497eC509, 1956
Carlson LA: Determination of serum triglycerides. J Atheroscler Res 37:334eC336, 1963
Ricquier D, Bouillaud F, Toumelin P, Mory G, Bazin R, Arch J, Penicaud L: Expression of uncoupling protein mRNA in thermogenic or weakly thermogenic brown adipose tissue: evidence for a rapid beta-adrenoreceptor-mediated and transcriptionally regulated step during activation of thermogenesis. J Biol Chem 261:13905eC13910, 1986
Sainsbury A, Baldock PA, Schwarzer C, Ueno N, Enriquez RF, Couzens M, Inui A, Herzog H, Gardiner EM: Synergistic effects of Y2 and Y4 receptors on adiposity and bone mass revealed in double knockout mice. Mol Cell Biol 23:5225eC5233, 2003
Colombo C, Arlati S, Curcio L, Maiavacca R, Garatti M, Ronchi M, Corbetta C, Giunta A: Effect of taurine supplementation on fat and bile acid absorption in patients with cystic fibrosis. Scand J Gastroenterol Suppl 143:151eC156, 1988
Furuse M, Mabayo RT, Okumura J: Effects of dietary cholestyramine on the utilisation of diets containing medium or long chain triacylglycerols by chicks. Br Poult Sci 38:436eC438, 1997
Hoentjen F, Hopman WP, Jansen JB: Effect of circulating peptide YY on gallbladder emptying in humans. Scand J Gastroenterol 36:1086eC1091, 2001
Adrian TE, Mitchenere P, Sagor G, Bloom SR: Effect of pancreatic polypeptide on gallbladder pressure and hepatic bile secretion. Am J Physiol 243:G204eCG207, 1982
Pomeranz IS, Davison JS, Shaffer EA: In vitro effects of pancreatic polypeptide and motilin on contractility of human gallbladder. Dig Dis Sci 28:539eC544, 1983
Satchithanandam S, Vargofcak-Apker M, Calvert RJ, Leeds AR, Cassidy MM: Alteration of gastrointestinal mucin by fiber feeding in rats. J Nutr 120:1179eC1184, 1990
Pettersson M, Ahreen B, Lundquist I, Bttcher G, Sundler F: Neuropeptide Y: intrapancreatic neuronal localization and effects on insulin secretion in the mouse. Cell Tissue Res 248:43eC48, 1987
Moltz JH, McDonald JK: Neuropeptide Y: direct and indirect action on insulin secretion in the rat. Peptides 6:1155eC1159, 1985
Morgan DG, Kulkarni RN, Hurley JD, Wang ZL, Wang RM, Ghatei MA, Karlsen AE, Bloom SR, Smith DM: Inhibition of glucose stimulated insulin secretion by neuropeptide Y is mediated via the Y1 receptor and inhibition of adenylyl cyclase in RIN 5AH rat insulinoma cells. Diabetologia 41:1482eC1491, 1998
Cho YR, Kim CW: Neuropeptide Y promotes beta-cell replication via extracellular signal-regulated kinase activation. Biochem Biophys Res Commun 314:773eC780, 2004
Standridge M, Alemzadeh R, Zemel M, Koontz J, Moustaid-Moussa N: Diazoxide down-regulates leptin and lipid metabolizing enzymes in adipose tissue of Zucker rats. FASEB J 14:455eC460, 2000
Burcelin R, Brunner H, Seydoux J, Thorensa BTP: Increased insulin concentrations and glucose storage in neuropeptide Y Y1 receptor-deficient mice. Peptides 22:421eC427, 2001
Onaga T, Zabielski R, Kato S: Multiple regulation of peptide YY secretion in the digestive tract. Peptides 23:279eC290, 2002
Imamura M: Effects of surgical manipulation of the intestine on peptide YY and its physiology. Peptides 23:403eC407, 2002
Challis BG, Pinnock SB, Coll AP, Carter RN, Dickson SL, O’Rahilly S: Acute effects of peptide YY3eC36 on food intake and hypothalamic neuropeptide expression in the mouse. Biochem Biophys Res Commun 311:915eC919, 2003
Stephens TW, Basinski M, Bristow PK, Bue-Valleskey JM, Burgett SG, Craft L, Hale J, Hoffmann J, Hsiung HM, Kriauciunas A, MacKellar W, Rosteck PR, Schoner B, Smith D, Tinsley FC, Zhang XY, Heiman M: The role of neuropeptide Y in the antiobesity action of the obese gene product. Nature 377:530eC532, 1995
Guan XM, Yu H, Van der Ploeg LH: Evidence of altered hypothalamic pro-opiomelanocortin/ neuropeptide Y mRNA expression in tubby mice. Brain Res Mol Brain Res 59:273eC279, 1998
Mizuno TM, Kleopoulos SP, Bergen HT, Roberts JL, Priest CA, Mobbs CV: Hypothalamic pro-opiomelanocortin mRNA is reduced by fasting and [corrected] in ob/ob and db/db mice, but is stimulated by leptin. Diabetes 47:294eC297, 1998
Huang XF, Han M, South T, Storlien L: Altered levels of POMC, AgRP and MC4-R mRNA expression in the hypothalamus and other parts of the limbic system of mice prone or resistant to chronic high-energy diet-induced obesity. Brain Res 992:9eC19, 2003(Amanda Sainsbury, Hugo T.)