Objective:

To further address the function of the Y5 receptor in energy homeostasis, we investigated the effects of a novel spironolactone Y5 antagonist in diet-induced obese (DIO) mice.

Methods and Procedures:

Male C57BL/6 or Npy5r^–/– mice were adapted to high-fat (HF) diet for 6–10 months and were submitted to three experimental treatments. First, the Y5 antagonist at a dose of 10 or 30 mg/kg was administered for 1 month to DIO C57BL/6 or Npy5r^–/– mice. Second, the Y5 antagonist at 30 mg/kg was administered for 1.5 months to DIO C57BL/6 mice, and insulin sensitivity was evaluated using an insulin tolerance test. After a recovery period, nuclear magnetic resonance measurement was performed to evaluate body composition. Third, DIO mice were treated with the Y5 antagonist alone, or in combination with 10% food restriction, or with another anorectic agent, sibutramine at 10 mg/kg, for 1.5 months. Plasma glucose, insulin, and leptin levels, and adipose tissue weights were quantified.

Results:

The spironolactone Y5 antagonist significantly reduced body weight in C57BL DIO mice, but not in Npy5r^–/– DIO mice. The Y5 antagonist produced a fat-selective loss of body weight, and ameliorated obesity-associated insulin resistance in DIO mice. In addition, the Y5 antagonist combined with either food restriction or sibutramine tended to produce greater body weight loss, as compared with single treatment.

Discussion:

These findings demonstrate that the Y5 receptor is an important mediator of energy homeostasis in rodents.

Introduction

Obesity is associated with an increased risk of type 2 dependent diabetes and cardiovascular disease, while weight loss of 5–10% has been shown to ameliorate these obesity-related comorbidities (1). Current obesity treatments involve lifestyle modification (diet and exercise), which rarely meets the patient needs, and often additional pharmacotherapy. Only two drugs, sibutramine and orlistat, are approved for long-term use with diet and exercise, and both medications produce modest weight loss between 5 and 10% (2); however, the drug-induced weight loss is only 2–4 kg greater than that produced by placebo (3), and because lifestyle modifications are usually transient, patients often experience eventual weight regain (4). Therefore, additional therapeutic options are needed for treating obesity, and numerous regulators of food intake have been explored as targets for novel therapies. Neuropeptide Y (NPY), a 36-amino acid peptide neurotransmitter (5), is one of the most potent orexigenic peptides, and it is involved in several aspects of energy homeostasis. NPY actions are mediated through at least five receptors, Y1, Y2, Y4, Y5, and mY6, of which Y1, Y2, and Y5 are abundantly expressed in the hypothalamus (6). Y1 and Y5 selective agonists stimulate feeding (7,8,9,10), while Y2 selective agonists inhibit food intake (11). Therefore, Y1 or Y5 receptor antagonists, or Y2 receptor agonists may represent new options for treating obesity.

We recently found that a selective Y5 antagonist produced antiobesity effects in diet-induced obese (DIO) animals and the mechanism-based action of the Y5 antagonist was confirmed using Npy5r^–/– mice (12). However, Npy5r^–/– mice exhibit a mild obesity (13), and there are several conflicting reports regarding the antiobesity effects of Y5 antagonists. Two Y5 antagonists, CGP71683A and GW438014A, suppressed body weight gain in DIO and genetically obese models, whereas Y5 antagonist NPY5RA-972 had no effect in DIO rats (14,15,16). Therefore, evaluation of a structurally diverse Y5 antagonist in wild-type and Npy5r^–/– mice is important to confirm the role of the Y5 receptor in energy homeostasis. In this study, we evaluated the antiobesity effects of a structurally novel spironolactone Y5 antagonist in wild-type and Npy5r^–/– DIO mice in order to clarify the involvement of the Y5 receptor in obesity. We also conducted combination treatments with food restriction and sibutramine to further evaluate the potential of a Y5 antagonist as an antiobesity agent.

Methods and Procedures

Materials

[125I]PYY and [125I]PP were obtained from NEN Life Science Products, Perkin Elmer (Boston, MA). Culture reagents were from Life Science Technologies (Grand Island, NY). All other chemicals were of analytical grade. Sibutramine was purchased from ChemPacific (Baltimore, MD). The Y5 antagonist 3-oxo-N-(5-phenylpyrazinyl) spiro (isobenzofuran-1(3H), 4'-piperidine)-1'-carboxamide was synthesized by Banyu Pharmaceutical (Tsukuba, Japan, Figure 1).

Figure 1.

Structure of the spironolactone Y5 antagonist.

Full figure and legend (4K)

Expression of NPY receptors in COS-7 cells and receptor binding assays

The coding regions of human (h) Y1, hY2, hY4, and hY5, and mouse (m) Y5 genes were cloned by PCR into the multiple cloning site of pCI-neo (Promega, Madison, WI) with an optimal Kozac sequence, GCCGCCACC, before the ATG start codons. Nucleotide sequences of the resulting clones were confirmed to be free of PCR-induced errors using an ABI 373A automated sequencer (Perkin Elmer, Norwalk, CT). After DNA was transfected into COS-7 cells, membranes were prepared, binding reactions were performed, and the data were analyzed as described previously (17), except that artificial cerebrospinal fluid buffer (Life Technologies, Bethesda, MD) was used as the binding buffer. Artificial cerebrospinal fluid buffer contained 1.5 mmol/l CaCl₂, 4 mmol/l KCl, 120 mmol/l NaCl, 1 mmol/l mol/lgCl₂, and 25 mmol/l NaHCO₃.

Measurement of intracellular calcium ion concentrations

[Ca²⁺]_i was measured fluorometrically using a Ca²⁺-sensitive fluorescent dye, fura-2. Cells expressing human NPY receptors were harvested using 0.25% trypsin and 0.02% EDTA. Cells (1.0 10⁷ cells) were washed once with Krebs-Henseleit HEPES buffer containing 0.1% bovine serum albumin (pH 7.4), suspended in 1 ml of buffer, and incubated with 2 mmol/l fura-2 acetoxymethyl ester at 37 °C for 60 min. Fura-2-loaded cells were washed with buffer and resuspended in 10 ml of buffer. The resultant suspension (0.5 ml) was stirred continuously at 37 °C in a cuvette during measurement. The test compound or vehicle was added 5 min before adding NPY, and fluorescent intensity at an emission wavelength of 500 nm and excitation wavelengths of 340 and 380 nm was monitored using a CAF-110 intracellular ion analyzer (Jasco, Tokyo, Japan). [Ca²⁺]_i values were calculated as reported previously (18).

Animals

Male C57BL/6J and C57BL/6N mice (age, 18 weeks; CLEA Japan, Tokyo, Japan) and male Npy5r^–/– mice were housed individually in plastic cages and were kept at 23 2 °C and 55 15% relative humidity on a 12-h light–dark cycle (7 PM, lights off). Generation of Npy5r^–/– mice was as described previously (19). Male Npy5r^–/– mice were backcrossed for six generations into a C57BL/6N background, and C57BL/6N mice were used as control mice. Mice were fed either a high-fat diet (HF diet; F3282; Bio-Serv, NJ) or moderately HF diet (MHF diet; Oriental Bioservice Kanto, Ibaraki, Japan) ad libitum for 6–10 months before drug treatment was initiated. The MHF diet provides 52.4% energy as carbohydrate, 15.0% as protein, and 32.6% as fat (4.4 kcal/g), whereas the HF diet provides 29.8% energy as carbohydrate, 16.0% as protein, and 54.2% as fat (5.3 kcal/g). All experimental procedures followed the Japanese Pharmacological Society Guidelines for Animal Use.

Procedure

Chronic treatment with the Y5 antagonist in Npy5r^–/– DIO mice. MHF diet-fed Npy5r^–/– or C57BL/6N mice were divided into three groups and each group was orally administered either the vehicle (0.5% methylcellulose) or the Y5 antagonist at a dose of 10 or 30 mg/kg once daily for 1 month. Drug administration was performed 1.5 h before the beginning of the dark period and after the measurement of daily food intake and body weight.

Chronic treatment with the Y5 antagonist in DIO mice. HF diet-fed C57BL/6J mice were divided into two groups matched for body weight, and each group was orally administered either the vehicle (0.5% methylcellulose) or the Y5 antagonist at 30 mg/kg once daily for 2 months. Administration was performed at 1.5 h before the beginning of the dark period and after the measurement of daily food intake and body weight.

After 6 weeks of administration, mice were fasted for 2 h, and insulin (0.75 U/kg) was injected intraperitoneally. Blood samples at 0, 15, 30, 60, 90, and 120 min were collected from the tail vein, and blood glucose was measured. After a 4-week recovery period, mice were fasted for 2 h and blood samples for leptin and insulin measurement were collected from the infraorbital veins, and thereafter nuclear magnetic resonance measurement (Minispec Fat/Lean mass analyzer mq10, Bruker Optics, Billerica, MA) was performed to evaluate body composition.

Combination treatment with food restriction or sibutramine in DIO mice. In order to investigate the combination of the Y5 antagonist with food restriction, MHF diet-fed C57BL/6J mice were divided into four groups: vehicle-treated/ad libitum-fed; vehicle-treated/10% food restriction; Y5 antagonist 30 mg/kg treated/ad libitum-fed; and Y5 antagonist 30 mg/kg treated/10% food restriction. Daily food intake in the food-restricted group was restricted to 90% of their average food intake during the pretreatment period, with 10 and 90% of the daily amount of food given to the mice at 8 AM and 6 PM, respectively. The Y5 antagonist was administered twice daily for 1.5 months at 1.5 h before the beginning of the dark period, and at 1.5 h after the beginning of the light period. Evening administration was performed after the measurement of daily food intake and body weight.

In order to investigate the combination of the Y5 antagonist with sibutramine, MHF diet-fed C57BL/6J mice were divided into four groups: vehicle alone; Y5 antagonist 30 mg/kg; sibutramine 10 mg/kg; and sibutramine 10 mg/kg/Y5 antagonist 30 mg/kg. Drug administration was performed at 1.5 h before the beginning of the dark period for 1.5 months.

After final drug administration, mice were fasted for 2 h, and blood samples were collected from the infraorbital vein for measurement of plasma glucose, insulin, and leptin levels. Under isoflurane anesthesia, mice were killed for collection of whole blood from the heart. Mesenteric adipose tissue was excised and weighed.

Measurement of hormone and blood chemistry

Plasma glucose was measured using a commercial kit (Determiner GL-E kit; Kyowa Medex, Tokyo, Japan). Insulin and leptin levels were measured by ELISA (Morinaga, Yokohama, Japan).

Statistical analysis

Data are expressed as means s.e.m. Body weight changes were compared between groups using repeated measure two-way ANOVA coupled with post hoc Bonferroni test. For food intake, blood parameters and tissue weights, one-way ANOVA coupled with post hoc Bonferroni test was performed. P <0.05 was considered significant.

Results

Selectivity and potency of the Y5 antagonist in NPY receptors

Specific binding of 125I-PYY to human and mouse Y5 receptors was potently inhibited by the spironolactone Y5 antagonist (K_i = 2.4 and 1.7 nmol/l, Table 1). In contrast, the Y5 antagonist showed low affinity for other cloned NPY receptors (Y1, Y2, and Y4 receptors). The Y5 antagonist dose-dependently inhibited the NPY-induced [Ca²⁺]_i increase with a K_b value of 3.6 nmol/l in CHO cells expressing human Y5 receptors (Table 1). The Y5 antagonist alone lacked agonist activity because it did not induce a [Ca²⁺]_i increase at 1 mol/l (data not shown).

Table 1 - Pharmacological profiles of Y5 antagonist for neuropeptide Y (NPY) receptors.

Full table (11K)

Chronic treatment of the Y5 antagonist in Npy5r^–/– DIO mice

At the start of drug administration, MHF diet-fed Npy5r^–/– mice weighed 50.3 0.4 g, while C57BL/6N mice weighed 47.1 0.5 g. As shown in Figure 2, the Y5 antagonist at 10 and 30 mg/kg had no effect on body weight in the Npy5r^–/– mice, while the Y5 antagonist significantly and dose-dependently reduced body weight in DIO C57BL/6N mice (P < 0.01). Thus, the Y5 antagonist acts via the Y5 receptor in a mechanism-dependent manner to cause body weight loss.

Figure 2.

Effects of the Y5 antagonist on body weight change in diet-induced obese of (a) wild type and (b) Y5 KO C57BL/6N mice. The Y5 antagonist was orally administered for 1 month at doses of 10 or 30 mg/kg. Values are means s.e. of five to seven mice per group. ^##P < 0.01 vs. vehicle-treated group. KO, knockout.

Full figure and legend (15K)

Chronic treatment with the Y5 antagonist in DIO mice

We evaluated the effect of the Y5 antagonist on body composition and obesity-related insulin resistance in DIO mice. At the start of drug administration, HF diet-fed C57BL/6J mice weighed 54.0 0.9 g, while regular chow-fed C57BL/6J mice weighed 34.1 0.4 g. The Y5 antagonist at 30 mg/kg significantly reduced body weight in the DIO mice (Figure 3a, P < 0.01). After 6 weeks of treatment, the body weight of the HF-diet fed, vehicle-treated mice had increased to 57.3 0.7 g, and that of regular chow-fed mice had increased to 35.2 0.5 g, but the body weight of Y5 antagonist treated mice had decreased to 52.3 0.8 g. Basal glucose levels were significantly increased in DIO mice when compared with those in lean mice (P < 0.01, 184.9 7.1 mg/dl vs. 149.1 9.5 mg/dl, respectively), but glucose levels were not affected by Y5 antagonist treatment (178.4 3.3 mg/dl). Insulin sensitivity evaluated using insulin tolerance test was significantly ameliorated through Y5 antagonist treatment (Figure 3b). At the end of treatment, the Y5 antagonist significantly decreased body fat content (P < 0.01), but did not affect lean mass content (Figure 4a,b). The Y5 antagonist significantly decreased plasma insulin levels (P < 0.01), which were elevated in the vehicle-treated DIO mice (Figure 4c, P < 0.01).

Figure 3.

Effects of the Y5 antagonist on (a) body weight changes and (b) insulin tolerance test in C57BL/6J diet-induced obese mice. The Y5 antagonist was orally administered for 1 month at 30 mg/kg. Values are means s.e. of 8–15 mice per group. ^#P < 0.05, ^##P < 0.01 vs. vehicle-treated high-fat diet (HFD)-fed group. RD, regular diet.

Full figure and legend (16K)

Figure 4.

Effects of the Y5 antagonist on body composition and plasma insulin level. The Y5 antagonist was orally administered for 1.5 months at 30 mg/kg, and (a) fat and (b) lean mass content were estimated by NMR measurement, and (c) plasma insulin level were measured. Values are means s.e. of 8–15 mice per group. ^##P < 0.01 vs. regular chow-fed group, *P < 0.05, **P < 0.01 vs. vehicle-treated HFD-fed group. RD, regular diet; HFD, high-fat diet.

Full figure and legend (11K)

Combination treatment with food restriction or sibutramine in DIO mice

In the combination study with food restriction, MHF diet-fed C57BL/6J mice weighed 49.0 0.9 g at the start of drug administration. The Y5 antagonist at 30 mg/kg twice daily, and 10% food restriction significantly reduced body weight in DIO mice, by 9 and 7%, respectively, relative to vehicle-treated mice (Figure 5a, P < 0.01). The Y5 antagonist mildly suppressed food intake, and decreased cumulative food intake by 6% (data not shown). Combined treatment with the Y5 antagonist, and food restriction produced a greater reduction in body weight than that observed with either treatment alone. At the end of administration of the Y5 antagonist and food restriction, the mice weighed 13% less than the vehicle-treated group. Mesenteric adipose tissue was significantly reduced by treatment with the Y5 antagonist alone and the combination treatment (P < 0.01), but was not significantly reduced by food restriction alone (Figure 5b).

Figure 5.

Combined effects of the Y5 antagonist with food restriction on (a) body weight changes and (b) mesenteric adipose tissue weight in C57BL6J diet-induced obese mice. Mice were fed ad libitum or restricted to 90% of their basal food intake, and administered the vehicle or the Y5 antagonist at 30 mg/kg twice daily for 1.5 months. Values are means s.e. of 6–10 mice per group. ^##P < 0.01 vs. vehicle-treated group.

Full figure and legend (17K)

In the combination study with sibutramine, MHF diet-fed C57BL/6J mice weighed 55.7 0.6 g at the start of drug administration. The Y5 antagonist at 30 mg/kg, and sibutramine at 10 mg/kg, significantly reduced body weight in the DIO mice (Figure 6a, P < 0.01). The body weight difference relative to vehicle-treated mice reached -9.4 and -8.4%, respectively. Sibutramine transiently and potently suppressed food intake, and thereafter, feeding suppression gradually recovered to the level of the vehicle-treated group (Figure 6b). Combination treatment with the Y5 antagonist and sibutramine produced greater body weight loss when compared with either single treatment. At the end of the experiment, the combination-treated mice weighed 12.7% less than the vehicle-treated group. Mesenteric adipose tissue was significantly reduced by treatment with the Y5 antagonist and the combination treatment (P < 0.01), but was not significantly reduced by treatment with sibutramine alone (Figure 6c).

Figure 6.

Combined effects of the Y5 antagonist with sibutramine on (a) body weight changes, (b) food intake, and (c) mesenteric adipose tissue weight in C57BL/6J diet-induced obese mice. Mice were administered either the vehicle or the Y5 antagonist at 30 mg/kg or sibutramine at 10 mg/kg alone, or both the Y5 antagonist and sibutramine for 1.5 months. Values are means s.e. of five to eight mice per group. ^##P < 0.01 vs. vehicle-treated group.

Full figure and legend (20K)

Discussion

In this study, we evaluated the antiobesity effects of a novel, spironolactone Y5 antagonist in DIO mice in order to further clarify the involvement of Y5 receptors in obesity development, and to refine the antiobesity profile of the Y5 antagonist. We previously reported that a xanthen class Y5 antagonist produced antiobesity effects only in wild-type DIO animals, and suggested that the Y5 receptor might be involved in diet-induced obesity development (12). However, because Npy5r^–/– mice exhibit a mildly obese phenotype rather than a lean phenotype (13), and because there are several conflicting reports regarding the antiobesity effects of Y5 antagonists (14,15,16), the function of the Y5 receptor in obesity development is controversial. Thus, evaluation of structurally diverse compounds is important to clarify the role of the Y5 receptor in obesity. The spironolactone Y5 antagonist, which exhibits high selectivity for both the human and mouse Y5 receptors and potently blocks NPY action in human Y5 receptor–expressing cells, produced an antiobesity effect in C57BL/6 DIO mice, but not in Npy5r^–/– DIO mice. With regard to antiobesity profiles, we confirmed that the body weight loss produced by the Y5 antagonist was mainly due to the loss of fat mass, rather than the loss of lean mass, as estimated by nuclear magnetic resonance measurement. The Y5 antagonist had almost no effect on lean mass. In agreement with the reduction in adiposity, plasma leptin levels decreased significantly (data not shown). We also evaluated insulin sensitivity using an insulin tolerance test. It is known that insulin resistance is closely associated with obesity, and that insulin resistance can lead to various obesity-related comorbidities, such as type 2 diabetes and cardiovascular disease. The chronic treatment with the Y5 antagonist showed significant amelioration of insulin resistance in the insulin tolerance test. Because insulin sensitivity is negatively correlated with adiposity, the amelioration of insulin sensitivity is likely to be due to weight loss after chronic treatment. The data obtained here with the structurally diverse Y5 spironolactone, and the previous xanthen class Y5 antagonist (12), strongly supports a significant role for the Y5 receptor in energy homeostasis.

Generally, pharmacotherapy prescribed for human obesity includes a diet and exercise regimen, and produces a modest weight loss of between 5 and 10% (2). The antiobesity mechanism of a Y5 antagonist has been shown to involve both feeding suppression and prevention of the reduction in energy expenditure induced by feeding suppression (20). Thus, we hypothesize that continuous feeding suppression would prolong and increases the body weight reduction by a Y5 antagonist, which would result in a greater antiobesity effect. We conducted combination treatment with both the Y5 antagonist and food restriction in DIO mice to test this hypothesis. Food restriction reduced body weight, but the weight loss was less than that with the Y5 antagonist, even though the cumulative feeding suppression was greater than the feeding suppression effects of the Y5 antagonist. In addition, food restriction did not significantly reduce adipose tissue weight, nor did it affect the plasma insulin level (data not shown). These findings further support our previous observation that the Y5 antagonist affects both food intake and energy expenditure (20).

We have also reported that the Y5 antagonist increased uncoupling proteins and 3 adrenergic receptor mRNA levels in white adipose tissue, and decreased SREBP-1c mRNA level in liver, while pair-feeding treatment, such as food restriction treatment, did not affect any of these mRNA changes. Thus, the Y5 antagonist preferentially reduces adipose weight, while food restriction reduces both adipose and lean mass. The combination treatment prolonged and enhanced the weight loss effects when compared with either treatment alone. However, in this study, we were not able to detect significant changes in adipose tissue weight or plasma parameters (data not shown) in the combination treatment group, as compared with either of the single treatment group. Perhaps extending the treatment period would produce significant changes in these parameters.

To date, several synergistic or additive anorexigenic effects have been reported in rodents with various pharmacological combinations, such as opioid and cannabinoid antagonists (21), cholecystokinin and leptin (22), and glucagon-like peptide-1 agonist and PYY3-36 (23). In addition, combination therapy has been used to achieve greater weight loss efficacy in humans (24,25,26). Combined treatment with fenfluramine and phentermine, known as "fen-phen," was a popular treatment for human obesity in the 1990s, until severe side effects curtailed its use (27). Here, we conducted a combination treatment using the Y5 antagonist and another antiobesity agent, sibutramine. Sibutramine is a centrally acting antiobesity agent, a noradrenergic and serotonergic reuptake inhibitor (28). In a preliminary experiment, we confirmed that sibutramine dose-dependently suppressed food intake and reduced body weight in the DIO mice model, and the maximum effective dose of sibutramine was 10 mg/kg (data not shown). Sibutramine at 10 mg/kg produced potent feeding suppression and reduced body weight in this study. At the end of treatment, the body weight reduction by the combination of the Y5 antagonist, and sibutramine was numerically greater than that obtained with the Y5 antagonist or with the sibutramine alone. However, the kinetics of weight loss were different between the Y5 antagonist and sibutramine. Sibutramine potently reduced body weight initially, and later body weight seemed to rebound slightly, while the Y5 antagonist reduced body weight gradually during the treatment period. At least in part, this might reflect the difference in the anorexigenic efficacy of both agents, the Y5 antagonist reduced food intake slightly and continuously, while sibutramine potently reduced food intake only for a few days. Thus, in sibutramine-treated mice, energy expenditure might be reduced to compensate for the acute reduction of energy intake. Subsequently, energy intake may rebound to exceed energy expenditure leading to a slight regain in body weight. Combined treatment with the Y5 antagonist and the sibutramine produced the greatest weight loss, and prevented the body weight regain observed with sibutramine treatment alone. As in the study above where the Y5 antagonist was combined with food restriction, extending the treatment period is necessary for significant changes to be seen in these parameters.

In summary, we confirmed that a novel spironolactone Y5 antagonist has potent antiobesity effects in DIO mice. The results here agree with our previous findings using a structurally distinct Y5 antagonist (12). Both compounds act via the Y5 receptor, as neither had any effect in Npy5r^–/– DIO mice. Thus, it is certain that the NPY Y5 receptor pathway is involved in rodent energy homeostasis. In addition, combination treatments of Y5 antagonist with food restriction or the antiobesity agent sibutramine, produced greater weight loss than either single treatment alone, suggesting a potential use of a Y5 antagonist as an antiobesity agent. However, recent clinical trials have shown that a NPY Y5 antagonist has insufficient efficacy as monotherapy, or in combination therapy with orlistat or sibutramine, to be clinically useful. Erondu et al. demonstrated that the NPY Y5 antagonist MK-0557 (an analog of the spironolactone Y5 antagonist used in this study) produced only 1.6 kg placebo-subtracted weight loss after 52 weeks of treatment (29) and blockade of the NPY5R with MK-0557 did not increase the weight loss efficacy of either orlistat or sibutramine (30). Thus, it appears that rodents are more sensitive to Y5 antagonist treatment than humans.

Disclosure

The authors declared no conflict of interest

References

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