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high-sucrose-fed rats
Pharmacological Research 59 (2009) 248–253
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NEU-P11, a novel melatonin agonist, inhibits weight gain and improves insulin sensitivity in high-fat/high-sucrose-fed rats Meihua She a,b , Xiaojian Deng a , Zhenyu Guo a , Moshe Laudon c , Zhuowei Hu d , Duanfang Liao e , Xiaobo Hu b , Yi Luo b , Qingyun Shen b , Zehong Su b , Weidong Yin a,b,∗ a
Institute of Cardiovascular Disease, Key Laboratory for Arteriosclerology of Hunan Province, University of South China, Hengyang, China Department of Biochemistry and Molecular Biology, School of Life Sciences and Technology, University of South China, Hengyang, China Drug Discovery, Neurim Pharmaceuticals Ltd., Tel-Aviv, Israel d Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China e Institute of Pharmacy and Pharmacology, School of Life Sciences and Technology, University of South China, Hengyang, China b c
a r t i c l e
i n f o
Article history: Received 23 November 2008 Received in revised form 8 January 2009 Accepted 15 January 2009 Keywords: NEU-P11 Melatonin Agonist Body weight Insulin sensitivity High-fat/high-sucrose-fed
a b s t r a c t Evidences indicate that a complex relationship exists among sleep disorders, obesity and insulin resistance. NEU-P11 is a novel melatonin agonist used in treatment of psychophysiological insomnia, and in animal studies NEU-P11 showed sleep-promoting effect. In this study, we applied NEU-P11 on obese rats to assess its potential melatoninergic effects in vivo. Obese models were established using high-fat/highsucrose-fed for 5 months. NEU-P11 (10 mg/kg)/melatonin (4 mg/kg)/vehicle were administered by a daily intraperitoneal injection respectively for 8 weeks. Our results showed that NEU-P11 or melatonin inhibited both body weight gain and deposit of abdominal fat with no influence on food intake. The impaired insulin sensitivity and antioxidative potency were improved and the levels of plasma glucose, total cholesterol (TC), triglycerides (TG) decreased with an increased in HDL-cholesterol (HDL-c) after NEU-P11 or melatonin administration. These data suggest that NEU-P11, like melatonin, decreased body weight gain and improved insulin sensitivity and metabolic profiles in obese rats. We conclude that NEU-P11 has a melatoninergic effect on regulating body weight in obese rats and also improving metabolic profiles and efficiently enhancing insulin sensitivity. © 2009 Elsevier Ltd. All rights reserved.
1. Introduction Obesity is reaching epidemic proportions worldwide and is increasing at an alarming rate. This is being driven, at least in part, by the westernization of diet with high-fat and higher caloric intake [1]. Obesity related disorders, such as insulin resistance, type 2 diabetes, hypertension and atherosclerosis diseases, are placing a considerable strain on our healthcare system [2–4]. Furthermore, recent evidence suggests an association between overweight, obesity and sleep disorders. For example, subjects with sleep disorders have an increased risk of obesity [5] and overweight and obese participants report sleeping less than subjects with a normal BMI [6]. On the other hand, overwhelming evidence supports the link between sleep disorder and risk of insulin resistance and diabetes [1,7–10]. On the base of these studies, Spiegel et al. [11] concludes correlation amongst weight gain,
∗ Corresponding author at: Institute of Cardiovascular Disease, Key Laboratory for Arteriosclerology of Hunan Province, University of South China, Hengyang 421001, China. Tel.: +86 734 8282554; fax: +86 734 8281618. E-mail address: [email protected] (W. Yin). 1043-6618/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.phrs.2009.01.005
insulin resistance, type 2 diabetes and sleep disorders in a recent review. The prevalence of obesity and obesity related metabolic and sleep disorders emphasizes the need for concerted efforts to prevent obesity rather than just treatment of its associated diseases, and there is a significant need for new drugs that effectively manage obesity, insulin resistance and sleep disorder while minimizing the risk of significant adverse effects. Melatonin, a neurohormone synthesized and secreted at night mainly by the pineal gland in vertebrates, is known for its sleeppromoting effects, which include shortening of sleep latency and lengthening of sleep duration [12]. On the other hand, several reports indicate that melatonin affects food intake, adiposity and BW in some species, although such effects are highly variable, including decrease in fat mass and BW in Siberian hamster [13], rats [14–17] and goldfish [18], increase in gray mouse lemurs [19] and raccoon dog [20] and no modifications in rat or in vole [21,22]. Melatonin also influences liver, kidney, and muscle energy contents of mammals [23,24]. Nocturnal pineal melatonin peak was shown to be decreased significantly in high-fat fed rats [25]. Taken overall, these data suggest that melatonin has a strong regulatory effect on carbohydrate/lipid metabolism and BW. The exact mechanism of
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action is not yet fully understood but a direct effect of melatonin on adipocytes and an indirect effect via the sympathetic nervous system have both been reported [26]. Thus, melatonin has been the focus for its applicability for sleep problems and BW regulation without undesirable side effects. Melatonin has not, however, received regulatory approval from the US FDA as a drug, because it can be sold freely as a food supplement. Consequently, there has been an active search for patentable melatonin receptor ligands in recent years. NEU-P11 has been developed as a melatonin agonist with high affinity to melatonin receptors intended for the treatment of psychophysiological insomnia. In animal studies NEU-P11 demonstrates sleep promoting without deleterious effects on memory [27,28]. The aim of this study was to investigate possible melatoninergic action of NEU-P11 on metabolism in obese rats induced by high-fat/high-sucrose diet (HFSD). We demonstrate NEU-P11 can regulate BW and improve metabolic profiles.
and 6:00 p.m.). The total period for establishing the obesity model was 20 weeks. The body weight of each rat was recorded twice a week. The institutional guidelines of Nanhua University for animal care and use were followed. The local animal ethics committee of Nanhua University approved the conduct of this experiment.
2. Materials and methods
All rats were weighed twice weekly, on Mondays and Fridays, between 9:00 and 11:00 a.m. These two weights were averaged to yield a weekly weight for each animal. Body length were measured for the BMI of each rat [BMI = BW (kg)/[length (m)]2 ].
2.1. Reagents NEU-P11 (C13 H16 N2 O4 ) and melatonin (C13 H16 N2 O2 ) were provided by Neurim Pharmaceuticals Ltd. (Tel-Aviv, Israel); both were dissolved in ethanol, then diluted in saline with the final ethanol concentration 0.01%. Sucrose was obtained from Liuzhou sugar Co. (Guangxi, China) and lard was obtained from Hengyang Meat Product Co. (Hunan, China). 2.2. Animals Sixty male Sprague–Dawley rats, 2 months of age, weighed 150–200 g, were obtained from the barrier unit at the Laboratory Animal Center of Nanhua University (Hunan, China). Animals were housed two per cage and given free access to water and a standard laboratory diet. All rats were subjected to alternate 12-h periods of dark and light (lights on 6:00 a.m.–6:00 p.m.). Temperature and humidity were maintained at 23 ◦ C and 40–60%, respectively. Rats were randomized assigned to two dietary groups: (1) normal diet group (n = 12), fed with standard chow; (2) high-fat and highsucrose diet group (HFSD group, n = 48), fed with 53% normal diet supplemented with 10% lard and 37% sucrose (the composition is shown in Table 1), which was similar to a “diabetogenic” diet [29]. The amount of daily fodder was equal, and all the rats were fed three times daily on a sharp feeding schedule (at 6:00 a.m., 12:00 a.m., Table 1 The ingredients and nutritive values of diets. Components
Control diet (%)
High-fat/high-sucrose diet (%)
Rice Wheat bran Soybean meal Cottonseed meal Colza meal Fish powder Bone powder Calcium bicarbonate 0.8 Salt Trace elements 0.5 Vitamins Pork lard Sucrose Nutrients Total energy (MJ kg−1 ) Metabolizable energy (MJ kg−1 ) Crude protein (%) Crude fat (%) Carbohydrates (%)
64.11 10.51 11.98 4 4 2 1.1 0.8 0.5 0.5 0.5
33.98 5.57 6.35 2.12 2.12 1 0.5
16.48 13.34 15.98 4.71 62.19
18.46 15.95 8.48 12.5 69.59
0.5 0.5 10 37
2.3. Study protocol After 20 weeks of feeding, rats were divided into four groups as follows: Group 1: normal control (n = 10 from the normal control diet group) treated with intraperitoneal injection of vehicle (5 ml); Groups 2, 3, 4: obese rats induced by HFSD treated with intraperitoneal injection of NEU-P11 (10 mg/kg), melatonin (4 mg/kg) or vehicle, respectively (n = 10). These treatments were administered for 8 weeks daily at 8:00 p.m. 2.4. Body weights (BW) and BMI
2.5. Food consumption and body temperature Each rat was provided with approximately 20 g chow. Food consumption was measured by subtracting the remained food before feeding on Mondays, Wednesdays, and Fridays. These three values were averaged as an index of daily consumption for the week. Relative food intake was calculated as grams of daily food intake per 100 g BW. A temperature probe (HP temperature module M 1029A) was inserted up to 1.5 cm in the rectum after induction of anesthesia and the rat rectal temperature was recorded. This was done three times every week at 7:00 a.m. and an average value was obtained as the body temperature 2.6. Measurement of plasma parameters Tail blood after fasting overnight was collected at the end of first 20 weeks and every 2 weeks after that. Plasma was separated by centrifugation (12,000 rpm, 4 min) and stored at −80 ◦ C. Glucose was determined by commercial enzymatic methods (test kits, Shanghai Rongsheng Biotech Inc., Shanghai, China). Insulin was determined by radioimmunoassay using Insulin Radioimmunology kits (China Institute of Atomic Research, Beijing, China). Plasma total cholesterol (TC-test kit), HDL-C (HDL-C-test kit), triglycerides (TG-test kit), superoxide dismutase (SOD-test kit), glutathion peroxidase (GSH-PX-test kit) and malondialdehyde (MDA-test kit) were determined by commercially available enzymatic methods (Shanghai Rongsheng Biotech Inc., Shanghai, China). 2.7. Oral glucose tolerance test (OGTT) and insulin sensitivity assay (IST) In order to analyze the glucose tolerance and insulin sensitivity, we conducted an oral glucose tolerance test and insulin tolerance tests every 2 weeks during the drug administration period. After an overnight fast, rats were given a glucose tolerance test with a bolus of glucose (2 g/kg, 10% solution) intragastrically. Blood samples were collected at 0, 30, 60, 90, and 120 min. Insulin sensitivity tests were performed after a 2-h fast. Animals were briefly removed from their cage for the tail bleeds and injected subcutaneously at the back of the neck with 0.5 units/kg insulin (Shanghai Biochemistry factory, Shanghai, China) and then returned. In the simplified insulin tolerance test, blood glucose is measured at 0 and 30 min
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after insulin injection. The 30-min glucose value is used as an indicator of insulin resistance. 2.8. Sacrifice and dissection Rats were sacrificed by decapitation at the end of 8th week. Blood was collected, and plasma was stored at −80 ◦ C. Retroperitoneal (including peritoneal) adipose tissues were dissected and immediately weighed. 2.9. Statistical analysis Data are expressed as the mean ± S.D. One-way ANOVA was used to analyze the significance of differences between mean values, and different groups were analyzed by analysis of variance using Duncan’s multiple range test. A P-value of less than 0.05 was considered statistically significant. 3. Results 3.1. Effect of treatments on body and fat weight At the first 20 weeks, i.e. before NEU-P11 or melatonin treatment, HFSD feeding resulted in a more rapid increase in BW; and at the week 0 (at this time point the treatment with NEU-P11 had not been started), HFSD rats were significantly heavier compared to the normal control group (386.00 ± 29.98 g vs. 307.00 ± 36.03 g of controls; P < 0.01) (Fig. 1A). However, supplementing NEU-P11 to those obese rats resulted in a reverse in tendency toward gaining more BW, and decreased the BW of the HFSD rats compared to HFSD control group (P < 0.01). Fig. 1B shows the mean BW of each group throughout the treatment period of 8 weeks. At week 8 the BW of rats administrated with NEU-P11 even reached a level comparable to the normal control, non-high-fat and high-sucrosefed animals. The BW gain was significantly increased in rats from HFSD group compared with rats from HFSD + NEU-P11 or melatonin groups (P < 0.01; Fig. 1C). The weight of retroperitoneal, epiploon, and mesenteric fats relative to BW (fat, % of BW) was significantly higher in rats from HFSD group compared with rats from normal and HFSD + NEU-P11 or melatonin groups (P < 0.01; Fig. 1D). NEUP11 decreased abdominal fat accumulation by 52.82%. The BMI of HFSD rats was approximately 7.09 kg/m2 , while the NEU-P11 treated group was 5.47 kg/m2 which is comparable to the normal control value of 5.42 kg/m2 . 3.2. Effect of treatments on caloric intake An important question is whether the decrease of BW and fat accumulation was the result of decreased caloric intake. To verify that NEU-P11 had no effect on energy intake, food consumption was monitored and caloric intake was obtained. The results showed there were no significant effects of NEU-P11 or melatonin treatment on relative food intake (grams of daily food intake per 100 g BW; Fig. 2A). Since the HFSD littermates were much heavier, this translated into a significantly reduced feed efficiency under NEUP11or melatonin compared with HFSD rats. Consistent with that, body temperature (Fig. 2B) was found to be significantly higher in the NEU-P11 treated group after 3 weeks of treatment (P < 0.01; HFSD + NEU-P11 vs. HFSD). 3.3. Effects of treatments on rat’s metabolic profile Fig. 3 shows the changes in plasma glucose, total cholesterol, HDL-C and triglyceride concentrations in the four groups during the NEU-P11 or melatonin treatments. At the week 0, i.e. after the 20 weeks of high-fat/high-sucrose feeding, the levels of plasma
Fig. 1. Effects of treatments on body weight and fat weight of rats on HFSD as described in Section 3. (A) A representing photographs of obese (HFSD) and normal rats. (B) Time course of weight changes during treatments. (C) BW gain at the end of the experiment. (D) The weight of retroperitoneal fats relative to BW. HFSD: high-fat/high-sucrose diet group; HFSD + NEU-P11: high-fat/high-sucrose diet with NEU-P11 (10 mg/kg) I.P., HFSD + MLT: high-fat/high-sucrose diet with melatonin (4 mg/kg) I.P., Control: normal chow with saline. Values are expressed as means ± S.D. (n = 10). *P < 0.01 vs. control.
glucose, total cholesterol and the triglyceride were significantly higher in the HFSD groups compared with rats fed with control diet (P < 0.05; HFSD vs. control). After 8 weeks of NEU-P11 or melatonin treatment, the plasma glucose levels in HFSD + NEUP11 or melatonin group had been efficiently suppressed compared with rats in HFSD group (5.10 ± 0.95 mmol/l, 5.02 ± 1.17 mmol/l vs. 6.38 ± 0.56 mmol/l) (P < 0.05; HFSD + NEU-P11 or melatonin vs. HFSD) and even reached the level of rats fed with control diet (5.31 ± 0.81 mmol/l). In addition, treatment with NEU-P11 resulted in a decrease in total cholesterol and triglyceride (33.1% and 28.4%, respectively) and an elevation in HDL-C levels (increase by 44.0%), which did not show in HFSD groups. Moreover, the effects of NEU-
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Fig. 2. Daily feed intake and body temperature over the 8 weeks of treatments as described in Section 3. (A) Daily feed intake. (B) Rectal temperature. Values are expressed as means ± S.D. (n = 10). *P < 0.01 vs. HFSD.
P11 on glucose levels and lipids were similar to that of melatonin performed in this study. 3.4. Effects of treatments on whole body glucose clearance and insulin sensitivity In order to analyze the effect of a chronic treatment with NEUP11 on glucose tolerance and insulin sensitivity, we performed the OGTT and IST after treatment with NEU-P11 or melatonin for 8 weeks. Fig. 4 shows the changes in plasma glucose and insulin concentrations after the glucose load. Rats from the HFSD group showed marked glucose intolerance compared with those from both the normal and the NEU-P11 or melatonin groups (Fig. 4A). The deficient glucose removal seen in the HFSD group may be caused by impairment of acute insulin secretion (absence of the first phase of insulin secretion) in response to the glucose load (Fig. 4B). Glucose tolerance is a function of glucose-stimulated insulin secretion, hepatic glucose output and tissue insulin sensitivity. The contribution of insulin sensitivity to the difference in glucose tolerance in rats from the four groups was explored by evaluating the clearance of plasma glucose as a function of time after insulin injection. This measure of a whole body insulin sensitivity can be conveniently expressed by the 30-min glucose value. Fig. 4C shows plasma glucose levels after a subcutaneous administration of insulin (0.5 units/kg BW). Insulin injection caused a decrease of glucose levels with respect to basal glucose values by about 34%, 38% and 38% in group NEU-P11, melatonin-treated and control group at 30 min, whereas the decrease of plasma glucose in HFSD group was significantly lower. This suggests that NEU-P11 or melatonin could improve insulin sensitivity in high-fat/high-sucrose induced obese rats. 3.5. Effects of treatments on markers of oxidative stress Table 2 shows the levels of MDA and activities of SOD and GSHPX enzyme after 8 weeks of treatment. Obesity rats induced by high-fat/high-sucrose diet showed significant decrease of SOD and GSH-PX enzyme activities and an increase of MDA levels when compared to control rats (P < 0.05). However, administration of NEU-P11
Fig. 3. Improvement of the metabolic profiles of HFSD rats by NEU-P11 and melatonin treatments as described in Section 3. (A) Circulating glucose levels measured before and after NEU-P11 for 8 weeks. (B) Plasma total cholesterol (TC). (C) HDLcholesterol (HDL-c). (D) Triglyceride concentrations (TG) levels. Values are expressed as means ± S.D. (n = 10). *P < 0.05 vs. control; # P < 0.05 vs. HFSD.
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increased by about 26.7 and 52.9% respectively, as a result of NEUP11 treatment compared with HFSD rats with saline. These effects of improvement in oxidative stress were even more dramatic after chronic treatment with melatonin. 4. Discussion
Fig. 4. Plasma glucose and insulin concentrations in OGTT and IST following NEUP11 and melatonin treatments as described in Section 3. Changes in plasma glucose (A) and insulin (B) concentrations after the glucose load test at the end of week 8. After an overnight fast, rats were treated with a bolus of glucose (2 g/kg, 10% solution) intragastrically at 10:00 a.m., 2 h after the drug injection. Blood samples were collected at 0, 30, 60, 90, and 120 min and glucose levels were determined. Insulin tolerance tests (C) were performed after a 2-h fast. Rats were injected subcutaneously with 0.5 units/kg insulin. Blood glucose is measured at 0 and 30 min after insulin injection. Values are expressed as means ± S.D. (n = 10). *P < 0.05 vs. control.
or melatonin to HFSD-feed rats significantly reversed the activities of these enzymes towards near normalcy. MDA levels were decreased significantly in NEU-P11 treatment group compared with HFSD group (decreased by 29.2%); GSH-PX and SOD levels were
Table 2 The levels or activities of MDA, SOD, GSH-PX in the plasma at the end of the study.
HFSD NEU-P11 (10 mg/kg) Melatonin (4 mg/kg) Control
MDA (nmol/ml)
GSH-PX (U/ml)
3.05 ± 0.44 2.16 ± 1.28b 1.79 ± 0.59b 1.91 ± 0.48
805.72 ± 107.65 1020.61 ± 239.10 1132.72 ± 168.32b 1182.27 ± 149.81
a
SOD (nU/ml) a
118.41 299.40 231.09 297.73
± ± ± ±
27.30a 26.23b 28.31b 28.89
Values are means ± S.D. for groups of 10 rats each. Values are given statistically significance at P < 0.05. a HFSD vs. control group. b NEU-P11/melatonin-treated groups vs. HFSD.
Studies have shown that long-term imbalance between intake and expenditure of fat is a central factor in the etiology of obesity [30]. Our current food supply is high in fat and sugar. We hypothesized that HFS diet promoted obesity by increasing energy intake, thus increasing the probability of positive energy balance and weight gain, ultimately induced obesity. In this study, all the rats were given the same amount of diet. The results show that feeding of HFS diet to rats for 5 months induced obesity, and its related metabolic syndrome such as impaired glucose tolerance. As melatonin secretion increases soon after the onset of darkness and peaks in the middle of the night [31], NEU-P11 and melatonin were injected at 8:00 p.m. Our results showed that administration of NEU-P11 to the HFSD rats resulted in a reversed in the tendency toward gaining more BW, and decreased the BW of the rats compared to HFSD control group. At week 8 the BWs of rats administrated NEU-P11 even reached a level comparable to the normal control, non-high-fat and high-sucrose-fed animals. The NEU-P11 effect on BW was coinciding with that of melatonin in this study and other reports [16,17,26]. Furthermore, the BMI of HFSD rats (7.09 kg/m2 ) was significantly decreased by NEUP11 (5.47 kg/m2 ) and reached a level comparable to normal rats (5.42 kg/m2 ). These data suggested that NEU-P11 can effectively inhibit the BW gain at least in obese rats induced by the high calorie diet. The effect on BW was achieved despite the fact that NEU-P11, as well as melatonin, did not influence food intake apparently in the present experiment. This suggested that feed efficiency, a measure of metabolic function, was negative in the NEU-P11/melatonintreated rats and positive in HFSD controls. This effect of melatonin has been described in many reports [16,17,26,32]. However, there were also the opposite outcomes. Melatonin has been shown to decrease food intake in Siberian hamsters [13] and goldfish [33]. A recent report also claimed melatonin decreases BW of ovariectomized rats due to a lower food intake [34]. These inconsistencies may be explained by differences in species, age, and mode or time of melatonin treatment. In addition, NEU-P11 as well as melatonin increased daytime body temperature of obese rats in this study. Melatonin exhibits effect of increasing in physical activity and core body temperature of rats in research of Wolden-Hanson et al. [17]. In present study, great care was taken to minimize stress during the recording of body temperatures. We hypothesized that temperature changes may have been mediated by physical activity (though had not been accurately monitored). In any case, the higher body temperature explained larger energy expenditure in NEU-P11-treated rats. This, together with a lower feed efficiency, suggested changes in energy metabolism and in the ability to store excess energy as body fat and had been hypothesized to contribute to a decreased BW in NEU-P11-treated obese rats. On the contrary, the decreased body temperature and higher feed efficiency of control HFSD rats might be related to continual BW gain. Adipocytes synthesize and release a variety of factors associated with glucose and lipid metabolism [35]. In this study, large mass of fat tissue in the HFSD rats implied hypertrophic adipocytes which would generate or secrete more insulin resistance-related factors such as TNF-␣, which inhibits glucose uptake, and free fatty acids, which may inhibit glucose uptake and glucose use in the muscle. Therefore, glucose clearance and insulin sensitivity were decreased in HFSD rats and the plasma total cholesterol, triglyceride and glucose levels were higher compared to control rats.
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In addition, reduced antioxidant capacity and oxidative stress occur with increased adiposity and this represents a potential contribution to insulin resistance in obesity [36]. Our finding that obese rats have a lower total antioxidant capacity than normal rats is in accordance with the literature. Insulin resistance observed in pinealectomized rats suggests melatonin participate in response to insulin [37]. We observed that NEU-P11 as well as melatonin improved insulin intolerance in obese rats as verified by OGTT and IST. Furthermore, a reduction in plasma triglyceride, glucose and total cholesterol levels with concomitant elevation of HDL-C levels were observed. These results were coinciding with the data about melatonin [26,38]. At the same time, NEU-P11 and melatonin increased the activity of antioxidant enzymes and decreased the oxidative marker MDA. These data raised the possibility that a modulation of antioxidant enzymes and/or direct antioxidant effect of melatonin or NEU-P11 might improve the imbalance of reactive oxygen species production and capacity of antioxidant defenses observed in obese rats. We hypothesized, this, together with reduced mass of fat, could contribute to the improvement in insulin sensitivity in NEU-P11 treated rats. In summary, we demonstrated for the first time, to our best knowledge, the effects of NEU-P11, a novel melatonin agonist, on the HFSD-induced obesity in rats. Our results showed that NEUP11, as well as melatonin, inhibited BW gain and intraabdominal adiposity without altering food intake, improving insulin sensitivity and potential of antioxidation, decreasing plasma glucose, triglyceride and total cholesterol while increasing of HDL-c levels. These results suggested that NEU-P11, the agonist of melatonin, possess the effects of melatonin in regulating BW and improving metabolic profiles. We hypothesized it might substitute melatonin effectively in BW regulation and be potentially beneficial for the treatment of obesity related metabolic and sleep disorders since there are limitations on melatonin, such as variable effects on BW and adiposity [13] and being inhibited to apply as a drug. However, it is important to note that extrapolating from these findings to species other than the rat is not currently warranted, and what side effects of NEU-P11 will come along? Accordingly, the mechanisms by which NEU-P11 reverses obesity-associated metabolic changes are under further investigation. Acknowledgments The authors gratefully acknowledge financial support from the National Major Basic Research Program of China (973) (2006CB503808) and the National Natural Sciences Foundation of China (Projects 30370675 and 30470720). References [1] Nilsson PM, Roost M, Engstrom G, Hedblad B, Berglund G. Incidence of diabetes in middle-aged men is related to sleep disturbances. Diabetes Care 2004;27:2464–9. [2] Marx J. Unraveling the causes of diabetes. Science 2002;26:686–9. [3] Weyer C, Hanson K, Bogardus C, Pratley RE. Long-term changes in insulin action and insulin secretion associated with gain, loss, regain and maintenance of body weight. Diabetologia 2000;43:36–46. [4] Sentinelli F, Romeo S, Arca M, Filippi E, Leonetti F, Banchieri M, et al. Human resistin gene, obesity, and type 2 diabetes: mutation analysis and population study. Diabetes 2002;51:860–2. [5] Vioque J, Torres A, Quiles J. Time spent watching television, sleep duration and obesity in adults living in Valencia, Spain. Int J Obes Relat Metab Disord 2000;24:1683–8. [6] Vorona RD, Winn MP, Babineau TW, Eng BP, Feldman HR, Ware JC. Overweight and obese patients in a primary care population report less sleep than patients with a normal body mass index. Arch Intern Med 2005;165:25–30. [7] VanHelder T, Radomski MW. Sleep deprivation and the effect on exercise performance. Sports Med 1989;7:235–47. [8] Meisinger C, Heier M, Loewel H. The MONICA/KORA Augsburg Cohort Study Sleep disturbance as a predictor of type 2 diabetes mellitus in men and women from the general population. Diabetologia 2005;48:235–41.
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Pharmacological Research journal homepage: www.elsevier.com/locate/yphrs
NEU-P11, a novel melatonin agonist, inhibits weight gain and improves insulin sensitivity in high-fat/high-sucrose-fed rats Meihua She a,b , Xiaojian Deng a , Zhenyu Guo a , Moshe Laudon c , Zhuowei Hu d , Duanfang Liao e , Xiaobo Hu b , Yi Luo b , Qingyun Shen b , Zehong Su b , Weidong Yin a,b,∗ a
Institute of Cardiovascular Disease, Key Laboratory for Arteriosclerology of Hunan Province, University of South China, Hengyang, China Department of Biochemistry and Molecular Biology, School of Life Sciences and Technology, University of South China, Hengyang, China Drug Discovery, Neurim Pharmaceuticals Ltd., Tel-Aviv, Israel d Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China e Institute of Pharmacy and Pharmacology, School of Life Sciences and Technology, University of South China, Hengyang, China b c
a r t i c l e
i n f o
Article history: Received 23 November 2008 Received in revised form 8 January 2009 Accepted 15 January 2009 Keywords: NEU-P11 Melatonin Agonist Body weight Insulin sensitivity High-fat/high-sucrose-fed
a b s t r a c t Evidences indicate that a complex relationship exists among sleep disorders, obesity and insulin resistance. NEU-P11 is a novel melatonin agonist used in treatment of psychophysiological insomnia, and in animal studies NEU-P11 showed sleep-promoting effect. In this study, we applied NEU-P11 on obese rats to assess its potential melatoninergic effects in vivo. Obese models were established using high-fat/highsucrose-fed for 5 months. NEU-P11 (10 mg/kg)/melatonin (4 mg/kg)/vehicle were administered by a daily intraperitoneal injection respectively for 8 weeks. Our results showed that NEU-P11 or melatonin inhibited both body weight gain and deposit of abdominal fat with no influence on food intake. The impaired insulin sensitivity and antioxidative potency were improved and the levels of plasma glucose, total cholesterol (TC), triglycerides (TG) decreased with an increased in HDL-cholesterol (HDL-c) after NEU-P11 or melatonin administration. These data suggest that NEU-P11, like melatonin, decreased body weight gain and improved insulin sensitivity and metabolic profiles in obese rats. We conclude that NEU-P11 has a melatoninergic effect on regulating body weight in obese rats and also improving metabolic profiles and efficiently enhancing insulin sensitivity. © 2009 Elsevier Ltd. All rights reserved.
1. Introduction Obesity is reaching epidemic proportions worldwide and is increasing at an alarming rate. This is being driven, at least in part, by the westernization of diet with high-fat and higher caloric intake [1]. Obesity related disorders, such as insulin resistance, type 2 diabetes, hypertension and atherosclerosis diseases, are placing a considerable strain on our healthcare system [2–4]. Furthermore, recent evidence suggests an association between overweight, obesity and sleep disorders. For example, subjects with sleep disorders have an increased risk of obesity [5] and overweight and obese participants report sleeping less than subjects with a normal BMI [6]. On the other hand, overwhelming evidence supports the link between sleep disorder and risk of insulin resistance and diabetes [1,7–10]. On the base of these studies, Spiegel et al. [11] concludes correlation amongst weight gain,
∗ Corresponding author at: Institute of Cardiovascular Disease, Key Laboratory for Arteriosclerology of Hunan Province, University of South China, Hengyang 421001, China. Tel.: +86 734 8282554; fax: +86 734 8281618. E-mail address: [email protected] (W. Yin). 1043-6618/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.phrs.2009.01.005
insulin resistance, type 2 diabetes and sleep disorders in a recent review. The prevalence of obesity and obesity related metabolic and sleep disorders emphasizes the need for concerted efforts to prevent obesity rather than just treatment of its associated diseases, and there is a significant need for new drugs that effectively manage obesity, insulin resistance and sleep disorder while minimizing the risk of significant adverse effects. Melatonin, a neurohormone synthesized and secreted at night mainly by the pineal gland in vertebrates, is known for its sleeppromoting effects, which include shortening of sleep latency and lengthening of sleep duration [12]. On the other hand, several reports indicate that melatonin affects food intake, adiposity and BW in some species, although such effects are highly variable, including decrease in fat mass and BW in Siberian hamster [13], rats [14–17] and goldfish [18], increase in gray mouse lemurs [19] and raccoon dog [20] and no modifications in rat or in vole [21,22]. Melatonin also influences liver, kidney, and muscle energy contents of mammals [23,24]. Nocturnal pineal melatonin peak was shown to be decreased significantly in high-fat fed rats [25]. Taken overall, these data suggest that melatonin has a strong regulatory effect on carbohydrate/lipid metabolism and BW. The exact mechanism of
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action is not yet fully understood but a direct effect of melatonin on adipocytes and an indirect effect via the sympathetic nervous system have both been reported [26]. Thus, melatonin has been the focus for its applicability for sleep problems and BW regulation without undesirable side effects. Melatonin has not, however, received regulatory approval from the US FDA as a drug, because it can be sold freely as a food supplement. Consequently, there has been an active search for patentable melatonin receptor ligands in recent years. NEU-P11 has been developed as a melatonin agonist with high affinity to melatonin receptors intended for the treatment of psychophysiological insomnia. In animal studies NEU-P11 demonstrates sleep promoting without deleterious effects on memory [27,28]. The aim of this study was to investigate possible melatoninergic action of NEU-P11 on metabolism in obese rats induced by high-fat/high-sucrose diet (HFSD). We demonstrate NEU-P11 can regulate BW and improve metabolic profiles.
and 6:00 p.m.). The total period for establishing the obesity model was 20 weeks. The body weight of each rat was recorded twice a week. The institutional guidelines of Nanhua University for animal care and use were followed. The local animal ethics committee of Nanhua University approved the conduct of this experiment.
2. Materials and methods
All rats were weighed twice weekly, on Mondays and Fridays, between 9:00 and 11:00 a.m. These two weights were averaged to yield a weekly weight for each animal. Body length were measured for the BMI of each rat [BMI = BW (kg)/[length (m)]2 ].
2.1. Reagents NEU-P11 (C13 H16 N2 O4 ) and melatonin (C13 H16 N2 O2 ) were provided by Neurim Pharmaceuticals Ltd. (Tel-Aviv, Israel); both were dissolved in ethanol, then diluted in saline with the final ethanol concentration 0.01%. Sucrose was obtained from Liuzhou sugar Co. (Guangxi, China) and lard was obtained from Hengyang Meat Product Co. (Hunan, China). 2.2. Animals Sixty male Sprague–Dawley rats, 2 months of age, weighed 150–200 g, were obtained from the barrier unit at the Laboratory Animal Center of Nanhua University (Hunan, China). Animals were housed two per cage and given free access to water and a standard laboratory diet. All rats were subjected to alternate 12-h periods of dark and light (lights on 6:00 a.m.–6:00 p.m.). Temperature and humidity were maintained at 23 ◦ C and 40–60%, respectively. Rats were randomized assigned to two dietary groups: (1) normal diet group (n = 12), fed with standard chow; (2) high-fat and highsucrose diet group (HFSD group, n = 48), fed with 53% normal diet supplemented with 10% lard and 37% sucrose (the composition is shown in Table 1), which was similar to a “diabetogenic” diet [29]. The amount of daily fodder was equal, and all the rats were fed three times daily on a sharp feeding schedule (at 6:00 a.m., 12:00 a.m., Table 1 The ingredients and nutritive values of diets. Components
Control diet (%)
High-fat/high-sucrose diet (%)
Rice Wheat bran Soybean meal Cottonseed meal Colza meal Fish powder Bone powder Calcium bicarbonate 0.8 Salt Trace elements 0.5 Vitamins Pork lard Sucrose Nutrients Total energy (MJ kg−1 ) Metabolizable energy (MJ kg−1 ) Crude protein (%) Crude fat (%) Carbohydrates (%)
64.11 10.51 11.98 4 4 2 1.1 0.8 0.5 0.5 0.5
33.98 5.57 6.35 2.12 2.12 1 0.5
16.48 13.34 15.98 4.71 62.19
18.46 15.95 8.48 12.5 69.59
0.5 0.5 10 37
2.3. Study protocol After 20 weeks of feeding, rats were divided into four groups as follows: Group 1: normal control (n = 10 from the normal control diet group) treated with intraperitoneal injection of vehicle (5 ml); Groups 2, 3, 4: obese rats induced by HFSD treated with intraperitoneal injection of NEU-P11 (10 mg/kg), melatonin (4 mg/kg) or vehicle, respectively (n = 10). These treatments were administered for 8 weeks daily at 8:00 p.m. 2.4. Body weights (BW) and BMI
2.5. Food consumption and body temperature Each rat was provided with approximately 20 g chow. Food consumption was measured by subtracting the remained food before feeding on Mondays, Wednesdays, and Fridays. These three values were averaged as an index of daily consumption for the week. Relative food intake was calculated as grams of daily food intake per 100 g BW. A temperature probe (HP temperature module M 1029A) was inserted up to 1.5 cm in the rectum after induction of anesthesia and the rat rectal temperature was recorded. This was done three times every week at 7:00 a.m. and an average value was obtained as the body temperature 2.6. Measurement of plasma parameters Tail blood after fasting overnight was collected at the end of first 20 weeks and every 2 weeks after that. Plasma was separated by centrifugation (12,000 rpm, 4 min) and stored at −80 ◦ C. Glucose was determined by commercial enzymatic methods (test kits, Shanghai Rongsheng Biotech Inc., Shanghai, China). Insulin was determined by radioimmunoassay using Insulin Radioimmunology kits (China Institute of Atomic Research, Beijing, China). Plasma total cholesterol (TC-test kit), HDL-C (HDL-C-test kit), triglycerides (TG-test kit), superoxide dismutase (SOD-test kit), glutathion peroxidase (GSH-PX-test kit) and malondialdehyde (MDA-test kit) were determined by commercially available enzymatic methods (Shanghai Rongsheng Biotech Inc., Shanghai, China). 2.7. Oral glucose tolerance test (OGTT) and insulin sensitivity assay (IST) In order to analyze the glucose tolerance and insulin sensitivity, we conducted an oral glucose tolerance test and insulin tolerance tests every 2 weeks during the drug administration period. After an overnight fast, rats were given a glucose tolerance test with a bolus of glucose (2 g/kg, 10% solution) intragastrically. Blood samples were collected at 0, 30, 60, 90, and 120 min. Insulin sensitivity tests were performed after a 2-h fast. Animals were briefly removed from their cage for the tail bleeds and injected subcutaneously at the back of the neck with 0.5 units/kg insulin (Shanghai Biochemistry factory, Shanghai, China) and then returned. In the simplified insulin tolerance test, blood glucose is measured at 0 and 30 min
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after insulin injection. The 30-min glucose value is used as an indicator of insulin resistance. 2.8. Sacrifice and dissection Rats were sacrificed by decapitation at the end of 8th week. Blood was collected, and plasma was stored at −80 ◦ C. Retroperitoneal (including peritoneal) adipose tissues were dissected and immediately weighed. 2.9. Statistical analysis Data are expressed as the mean ± S.D. One-way ANOVA was used to analyze the significance of differences between mean values, and different groups were analyzed by analysis of variance using Duncan’s multiple range test. A P-value of less than 0.05 was considered statistically significant. 3. Results 3.1. Effect of treatments on body and fat weight At the first 20 weeks, i.e. before NEU-P11 or melatonin treatment, HFSD feeding resulted in a more rapid increase in BW; and at the week 0 (at this time point the treatment with NEU-P11 had not been started), HFSD rats were significantly heavier compared to the normal control group (386.00 ± 29.98 g vs. 307.00 ± 36.03 g of controls; P < 0.01) (Fig. 1A). However, supplementing NEU-P11 to those obese rats resulted in a reverse in tendency toward gaining more BW, and decreased the BW of the HFSD rats compared to HFSD control group (P < 0.01). Fig. 1B shows the mean BW of each group throughout the treatment period of 8 weeks. At week 8 the BW of rats administrated with NEU-P11 even reached a level comparable to the normal control, non-high-fat and high-sucrosefed animals. The BW gain was significantly increased in rats from HFSD group compared with rats from HFSD + NEU-P11 or melatonin groups (P < 0.01; Fig. 1C). The weight of retroperitoneal, epiploon, and mesenteric fats relative to BW (fat, % of BW) was significantly higher in rats from HFSD group compared with rats from normal and HFSD + NEU-P11 or melatonin groups (P < 0.01; Fig. 1D). NEUP11 decreased abdominal fat accumulation by 52.82%. The BMI of HFSD rats was approximately 7.09 kg/m2 , while the NEU-P11 treated group was 5.47 kg/m2 which is comparable to the normal control value of 5.42 kg/m2 . 3.2. Effect of treatments on caloric intake An important question is whether the decrease of BW and fat accumulation was the result of decreased caloric intake. To verify that NEU-P11 had no effect on energy intake, food consumption was monitored and caloric intake was obtained. The results showed there were no significant effects of NEU-P11 or melatonin treatment on relative food intake (grams of daily food intake per 100 g BW; Fig. 2A). Since the HFSD littermates were much heavier, this translated into a significantly reduced feed efficiency under NEUP11or melatonin compared with HFSD rats. Consistent with that, body temperature (Fig. 2B) was found to be significantly higher in the NEU-P11 treated group after 3 weeks of treatment (P < 0.01; HFSD + NEU-P11 vs. HFSD). 3.3. Effects of treatments on rat’s metabolic profile Fig. 3 shows the changes in plasma glucose, total cholesterol, HDL-C and triglyceride concentrations in the four groups during the NEU-P11 or melatonin treatments. At the week 0, i.e. after the 20 weeks of high-fat/high-sucrose feeding, the levels of plasma
Fig. 1. Effects of treatments on body weight and fat weight of rats on HFSD as described in Section 3. (A) A representing photographs of obese (HFSD) and normal rats. (B) Time course of weight changes during treatments. (C) BW gain at the end of the experiment. (D) The weight of retroperitoneal fats relative to BW. HFSD: high-fat/high-sucrose diet group; HFSD + NEU-P11: high-fat/high-sucrose diet with NEU-P11 (10 mg/kg) I.P., HFSD + MLT: high-fat/high-sucrose diet with melatonin (4 mg/kg) I.P., Control: normal chow with saline. Values are expressed as means ± S.D. (n = 10). *P < 0.01 vs. control.
glucose, total cholesterol and the triglyceride were significantly higher in the HFSD groups compared with rats fed with control diet (P < 0.05; HFSD vs. control). After 8 weeks of NEU-P11 or melatonin treatment, the plasma glucose levels in HFSD + NEUP11 or melatonin group had been efficiently suppressed compared with rats in HFSD group (5.10 ± 0.95 mmol/l, 5.02 ± 1.17 mmol/l vs. 6.38 ± 0.56 mmol/l) (P < 0.05; HFSD + NEU-P11 or melatonin vs. HFSD) and even reached the level of rats fed with control diet (5.31 ± 0.81 mmol/l). In addition, treatment with NEU-P11 resulted in a decrease in total cholesterol and triglyceride (33.1% and 28.4%, respectively) and an elevation in HDL-C levels (increase by 44.0%), which did not show in HFSD groups. Moreover, the effects of NEU-
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Fig. 2. Daily feed intake and body temperature over the 8 weeks of treatments as described in Section 3. (A) Daily feed intake. (B) Rectal temperature. Values are expressed as means ± S.D. (n = 10). *P < 0.01 vs. HFSD.
P11 on glucose levels and lipids were similar to that of melatonin performed in this study. 3.4. Effects of treatments on whole body glucose clearance and insulin sensitivity In order to analyze the effect of a chronic treatment with NEUP11 on glucose tolerance and insulin sensitivity, we performed the OGTT and IST after treatment with NEU-P11 or melatonin for 8 weeks. Fig. 4 shows the changes in plasma glucose and insulin concentrations after the glucose load. Rats from the HFSD group showed marked glucose intolerance compared with those from both the normal and the NEU-P11 or melatonin groups (Fig. 4A). The deficient glucose removal seen in the HFSD group may be caused by impairment of acute insulin secretion (absence of the first phase of insulin secretion) in response to the glucose load (Fig. 4B). Glucose tolerance is a function of glucose-stimulated insulin secretion, hepatic glucose output and tissue insulin sensitivity. The contribution of insulin sensitivity to the difference in glucose tolerance in rats from the four groups was explored by evaluating the clearance of plasma glucose as a function of time after insulin injection. This measure of a whole body insulin sensitivity can be conveniently expressed by the 30-min glucose value. Fig. 4C shows plasma glucose levels after a subcutaneous administration of insulin (0.5 units/kg BW). Insulin injection caused a decrease of glucose levels with respect to basal glucose values by about 34%, 38% and 38% in group NEU-P11, melatonin-treated and control group at 30 min, whereas the decrease of plasma glucose in HFSD group was significantly lower. This suggests that NEU-P11 or melatonin could improve insulin sensitivity in high-fat/high-sucrose induced obese rats. 3.5. Effects of treatments on markers of oxidative stress Table 2 shows the levels of MDA and activities of SOD and GSHPX enzyme after 8 weeks of treatment. Obesity rats induced by high-fat/high-sucrose diet showed significant decrease of SOD and GSH-PX enzyme activities and an increase of MDA levels when compared to control rats (P < 0.05). However, administration of NEU-P11
Fig. 3. Improvement of the metabolic profiles of HFSD rats by NEU-P11 and melatonin treatments as described in Section 3. (A) Circulating glucose levels measured before and after NEU-P11 for 8 weeks. (B) Plasma total cholesterol (TC). (C) HDLcholesterol (HDL-c). (D) Triglyceride concentrations (TG) levels. Values are expressed as means ± S.D. (n = 10). *P < 0.05 vs. control; # P < 0.05 vs. HFSD.
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increased by about 26.7 and 52.9% respectively, as a result of NEUP11 treatment compared with HFSD rats with saline. These effects of improvement in oxidative stress were even more dramatic after chronic treatment with melatonin. 4. Discussion
Fig. 4. Plasma glucose and insulin concentrations in OGTT and IST following NEUP11 and melatonin treatments as described in Section 3. Changes in plasma glucose (A) and insulin (B) concentrations after the glucose load test at the end of week 8. After an overnight fast, rats were treated with a bolus of glucose (2 g/kg, 10% solution) intragastrically at 10:00 a.m., 2 h after the drug injection. Blood samples were collected at 0, 30, 60, 90, and 120 min and glucose levels were determined. Insulin tolerance tests (C) were performed after a 2-h fast. Rats were injected subcutaneously with 0.5 units/kg insulin. Blood glucose is measured at 0 and 30 min after insulin injection. Values are expressed as means ± S.D. (n = 10). *P < 0.05 vs. control.
or melatonin to HFSD-feed rats significantly reversed the activities of these enzymes towards near normalcy. MDA levels were decreased significantly in NEU-P11 treatment group compared with HFSD group (decreased by 29.2%); GSH-PX and SOD levels were
Table 2 The levels or activities of MDA, SOD, GSH-PX in the plasma at the end of the study.
HFSD NEU-P11 (10 mg/kg) Melatonin (4 mg/kg) Control
MDA (nmol/ml)
GSH-PX (U/ml)
3.05 ± 0.44 2.16 ± 1.28b 1.79 ± 0.59b 1.91 ± 0.48
805.72 ± 107.65 1020.61 ± 239.10 1132.72 ± 168.32b 1182.27 ± 149.81
a
SOD (nU/ml) a
118.41 299.40 231.09 297.73
± ± ± ±
27.30a 26.23b 28.31b 28.89
Values are means ± S.D. for groups of 10 rats each. Values are given statistically significance at P < 0.05. a HFSD vs. control group. b NEU-P11/melatonin-treated groups vs. HFSD.
Studies have shown that long-term imbalance between intake and expenditure of fat is a central factor in the etiology of obesity [30]. Our current food supply is high in fat and sugar. We hypothesized that HFS diet promoted obesity by increasing energy intake, thus increasing the probability of positive energy balance and weight gain, ultimately induced obesity. In this study, all the rats were given the same amount of diet. The results show that feeding of HFS diet to rats for 5 months induced obesity, and its related metabolic syndrome such as impaired glucose tolerance. As melatonin secretion increases soon after the onset of darkness and peaks in the middle of the night [31], NEU-P11 and melatonin were injected at 8:00 p.m. Our results showed that administration of NEU-P11 to the HFSD rats resulted in a reversed in the tendency toward gaining more BW, and decreased the BW of the rats compared to HFSD control group. At week 8 the BWs of rats administrated NEU-P11 even reached a level comparable to the normal control, non-high-fat and high-sucrose-fed animals. The NEU-P11 effect on BW was coinciding with that of melatonin in this study and other reports [16,17,26]. Furthermore, the BMI of HFSD rats (7.09 kg/m2 ) was significantly decreased by NEUP11 (5.47 kg/m2 ) and reached a level comparable to normal rats (5.42 kg/m2 ). These data suggested that NEU-P11 can effectively inhibit the BW gain at least in obese rats induced by the high calorie diet. The effect on BW was achieved despite the fact that NEU-P11, as well as melatonin, did not influence food intake apparently in the present experiment. This suggested that feed efficiency, a measure of metabolic function, was negative in the NEU-P11/melatonintreated rats and positive in HFSD controls. This effect of melatonin has been described in many reports [16,17,26,32]. However, there were also the opposite outcomes. Melatonin has been shown to decrease food intake in Siberian hamsters [13] and goldfish [33]. A recent report also claimed melatonin decreases BW of ovariectomized rats due to a lower food intake [34]. These inconsistencies may be explained by differences in species, age, and mode or time of melatonin treatment. In addition, NEU-P11 as well as melatonin increased daytime body temperature of obese rats in this study. Melatonin exhibits effect of increasing in physical activity and core body temperature of rats in research of Wolden-Hanson et al. [17]. In present study, great care was taken to minimize stress during the recording of body temperatures. We hypothesized that temperature changes may have been mediated by physical activity (though had not been accurately monitored). In any case, the higher body temperature explained larger energy expenditure in NEU-P11-treated rats. This, together with a lower feed efficiency, suggested changes in energy metabolism and in the ability to store excess energy as body fat and had been hypothesized to contribute to a decreased BW in NEU-P11-treated obese rats. On the contrary, the decreased body temperature and higher feed efficiency of control HFSD rats might be related to continual BW gain. Adipocytes synthesize and release a variety of factors associated with glucose and lipid metabolism [35]. In this study, large mass of fat tissue in the HFSD rats implied hypertrophic adipocytes which would generate or secrete more insulin resistance-related factors such as TNF-␣, which inhibits glucose uptake, and free fatty acids, which may inhibit glucose uptake and glucose use in the muscle. Therefore, glucose clearance and insulin sensitivity were decreased in HFSD rats and the plasma total cholesterol, triglyceride and glucose levels were higher compared to control rats.
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In addition, reduced antioxidant capacity and oxidative stress occur with increased adiposity and this represents a potential contribution to insulin resistance in obesity [36]. Our finding that obese rats have a lower total antioxidant capacity than normal rats is in accordance with the literature. Insulin resistance observed in pinealectomized rats suggests melatonin participate in response to insulin [37]. We observed that NEU-P11 as well as melatonin improved insulin intolerance in obese rats as verified by OGTT and IST. Furthermore, a reduction in plasma triglyceride, glucose and total cholesterol levels with concomitant elevation of HDL-C levels were observed. These results were coinciding with the data about melatonin [26,38]. At the same time, NEU-P11 and melatonin increased the activity of antioxidant enzymes and decreased the oxidative marker MDA. These data raised the possibility that a modulation of antioxidant enzymes and/or direct antioxidant effect of melatonin or NEU-P11 might improve the imbalance of reactive oxygen species production and capacity of antioxidant defenses observed in obese rats. We hypothesized, this, together with reduced mass of fat, could contribute to the improvement in insulin sensitivity in NEU-P11 treated rats. In summary, we demonstrated for the first time, to our best knowledge, the effects of NEU-P11, a novel melatonin agonist, on the HFSD-induced obesity in rats. Our results showed that NEUP11, as well as melatonin, inhibited BW gain and intraabdominal adiposity without altering food intake, improving insulin sensitivity and potential of antioxidation, decreasing plasma glucose, triglyceride and total cholesterol while increasing of HDL-c levels. These results suggested that NEU-P11, the agonist of melatonin, possess the effects of melatonin in regulating BW and improving metabolic profiles. We hypothesized it might substitute melatonin effectively in BW regulation and be potentially beneficial for the treatment of obesity related metabolic and sleep disorders since there are limitations on melatonin, such as variable effects on BW and adiposity [13] and being inhibited to apply as a drug. However, it is important to note that extrapolating from these findings to species other than the rat is not currently warranted, and what side effects of NEU-P11 will come along? Accordingly, the mechanisms by which NEU-P11 reverses obesity-associated metabolic changes are under further investigation. Acknowledgments The authors gratefully acknowledge financial support from the National Major Basic Research Program of China (973) (2006CB503808) and the National Natural Sciences Foundation of China (Projects 30370675 and 30470720). References [1] Nilsson PM, Roost M, Engstrom G, Hedblad B, Berglund G. Incidence of diabetes in middle-aged men is related to sleep disturbances. Diabetes Care 2004;27:2464–9. [2] Marx J. Unraveling the causes of diabetes. Science 2002;26:686–9. [3] Weyer C, Hanson K, Bogardus C, Pratley RE. Long-term changes in insulin action and insulin secretion associated with gain, loss, regain and maintenance of body weight. Diabetologia 2000;43:36–46. [4] Sentinelli F, Romeo S, Arca M, Filippi E, Leonetti F, Banchieri M, et al. Human resistin gene, obesity, and type 2 diabetes: mutation analysis and population study. Diabetes 2002;51:860–2. [5] Vioque J, Torres A, Quiles J. Time spent watching television, sleep duration and obesity in adults living in Valencia, Spain. Int J Obes Relat Metab Disord 2000;24:1683–8. [6] Vorona RD, Winn MP, Babineau TW, Eng BP, Feldman HR, Ware JC. Overweight and obese patients in a primary care population report less sleep than patients with a normal body mass index. Arch Intern Med 2005;165:25–30. [7] VanHelder T, Radomski MW. Sleep deprivation and the effect on exercise performance. Sports Med 1989;7:235–47. [8] Meisinger C, Heier M, Loewel H. The MONICA/KORA Augsburg Cohort Study Sleep disturbance as a predictor of type 2 diabetes mellitus in men and women from the general population. Diabetologia 2005;48:235–41.
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