Comparison of independent and combined chronic metabolic effects of GIP and CB1 receptor blockade in high-fat fed mice

peptides 29 (2008) 1036–1041

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Comparison of independent and combined chronic metabolic effects of GIP and CB1 receptor blockade in high-fat fed mice Nigel Irwin *, Kerry Hunter, Peter R. Flatt School of Biomedical Sciences, University of Ulster, Coleraine, Northern Ireland BT52 1SA, UK

article info

abstract

Article history:

GIP receptor antagonism with (Pro3)GIP protects against obesity, insulin resistance, glucose

Received 13 November 2007

intolerance and associated disturbances in mice fed high-fat diet. Furthermore, cannabi-

Received in revised form

noid CB1 receptor antagonism with AM251 reduces appetite and body weight gain in mice.

8 January 2008

The present study has examined and compared the effects of chronic daily administrations

Accepted 11 January 2008

of (Pro3)GIP (25 nmol/kg body weight), AM251 (6 mg/kg body weight) and a combination of

Published on line 18 January 2008

both drugs in high-fat fed mice. Daily i.p. injection of (Pro3)GIP, AM251 or combined drug administration over 22 days significantly (P < 0.05 to <0.01) decreased body weight com-

Keywords:

pared with saline-treated controls. This was associated with a significant (P < 0.05 to <0.01)

Gastric inhibitory polypeptide (GIP)

reduction of food intake in mice treated with AM251. Plasma glucose levels and glucose

CB1 receptor

tolerance were significantly (P < 0.05) lowered by 22 days (Pro3)GIP, AM251 or combined drug

AM251

treatment. These changes were accompanied by a significant (P < 0.05) improvement of

Food intake

insulin sensitivity in all treatment groups. In contrast, AM251 lacked effects on glucose

Glucose homeostasis

tolerance, metabolic response to feeding and insulin sensitivity in high-fat mice when

High-fat feeding

administered acutely. These data indicate that chemical blockade of GIP- or CB1-receptor signaling using (Pro3)GIP or AM251, respectively provides an effective means of countering obesity and related abnormalities induced by consumption of high-fat energy-rich diet. AM251 lacks acute effects on glucose homeostasis and there was no evidence of a synergistic effect of combined treatment with (Pro3)GIP. # 2008 Elsevier Inc. All rights reserved.

1.

Introduction

Gastric inhibitory polypeptide (GIP) is a gut hormone secreted form the endocrine K-cells that regulates glucose homeostasis [11]. Despite its widely accepted role as a major insulinreleasing hormone of the enteroinsular axis, recent data indicate that GIP exerts important effects on lipid metabolism to promote fat deposition [26]. Thus, evidence is now accumulating to suggest a possible role for inhibition of GIP signaling in the alleviation of obesity [15,26,33]. Moreover, chronic daily administration of the stable and specific GIP receptor antagonist (Pro3)GIP [10], has been shown to prevent or even reverse many of the established metabolic abnormalities of genetic and dietary-induced obesity-diabetes [12,18,25].

Notwithstanding the clear potential of GIP receptor antagonism for alleviation of obesity and associated abnormalities [13,18] there is no evidence of reduced energy intake in animals with either genetic or chemical GIP receptor knockout [13,15]. Furthermore, studies have confirmed that GIP lacks any effect on feeding activity [32]. Thus, it may be possible to augment the beneficial effects of GIP receptor antagonism through a concurrent administration of therapy that reduces energy intake. Similarly, the actions of agents suppressing energy intake may be enhanced through parallel increases in energy expenditures. We have previously attempted to produce just such a scenario in high-fat fed mice through concurrent administration of peptide YY(3–36), (PYY(3–36)), with (Pro3)GIP [19]. However, we did not see a satiety effect of PYY(3–36) in

* Corresponding author. Tel.: +44 28 70 324313; fax: +44 28 70 324965. E-mail address: [email protected] (N. Irwin). 0196-9781/$ – see front matter # 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2008.01.006

peptides 29 (2008) 1036–1041

freely fed animals during a chronic treatment regime [19]. Thus, a second avenue for a restriction of energy intake in combination with (Pro3)GIP treatment is needed. The central endogenous cannabinoid system has been implicated in the control of appetite in humans and rodents [22,24]. This system comprises of two distinctly different receptor subtypes, CB1 and CB2 [17]. The major exogenous agonist of the CB1 receptor, D9-tetrehydrocannabinol causes marked hyperphagia in rodents [31] and man [9]. In keeping with this, genetic ablation of CB1 receptor signaling leads to leanness, resistance to diet-induced obesity and enhanced leptin sensitivity in mice [28]. Furthermore, chemical knockout of the CB1 receptor has significant anorectic effects in rats [1], mice [6] and monkeys [29]. Thus, current research is focusing on the potential of CB1 receptor antagonists for the alleviation of obesity through reduced energy intake [7]. AM251 (N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide) is a selective and potent CB1 receptor antagonist with well characterized anorectic effects in freely fed animals [16]. Our laboratory has previously shown an anorectic effect of daily chronic AM251 administration in obese diabetic (ob/ob) mice [20]. Thus, the present study was designed to evaluate the relative efficacy and combined activity of chronic treatment with two promising new classes of anti-obesity drugs, represented by (Pro3)GIP and AM251. This will address the possible beneficial effects of reduced energy intake by AM251 [16] in combination with increased energy expenditure through fat oxidation by blockade of GIP signaling [13,15]. Effects on body weight, food intake, basal plasma glucose and insulin, glucose homeostasis and insulin sensitivity were examined.

5% propylene glycol, 2% Tween 80), (Pro3)GIP (25 nmol/kg body weight; Sigma-Genosys Ltd., Cambridge, UK), AM251 (6 mg/kg body weight) or a combined drug administration at the same respective doses over a 22-day period. Food intake and body weight were recorded daily while non-fasting plasma glucose and insulin concentrations were monitored at 3–4-day intervals. Non-fasting intraperitoneal glucose tolerance (18 mmol/kg body weight) and insulin sensitivity (20 U/kg body weight) tests were performed at the end of the 22-day study period. In a separate series, high-fat fed mice not receiving treatment were used to examine possible acute effects of AM251 (6 mg/kg body weight) on intraperitoneal glucose tolerance (18 mmol/kg body weight), insulin sensitivity (20 U/kg body weight) and metabolic response to 15 min re-feeding. With the exception of insulin sensitivity tests, mice were fasted for 18 h prior to testing. All acute experiments commenced at 10:00 h. All animals tolerated the treatment regimes well and there were no obvious differences in behavior between the various groups of mice.

2.3.

Materials and methods

2.1.

Animals

Young female (8-week-old) NIH Swiss mice (Harlan U.K. Ltd.) were age-matched, divided into groups and housed individually in an air-conditioned room at 22  2 8C with a 12 h light:12 h dark cycle (08:00–20:00 h). Experimental animals had free access to drinking water and a high-fat diet (45% fat, 20% protein and 35% carbohydrate; percent of total energy of 26.15 kJ/g; Special Diets Service, Essex, UK). Prior to commencement of all experimental studies, animals were maintained on high-fat diet for 130 days. The generation of dietinduced obesity, insulin resistance and associated metabolic disturbances by mice fed this diet have been documented previously [25]. At the time of experimentation (130 days) obesity and diabetes were clearly manifest as judged by body weight and circulating plasma glucose levels. All animal experiments were carried out in accordance with the UK Animals (Scientific Procedures) Act 1986.

2.2.

Experimental protocols for in vivo studies

Groups of eight mice received once-daily intraperitoneal injections (17:00 h) of either saline vehicle (0.9% (w/v) NaCl,

Biochemical analysis

Blood samples taken from the cut tip of the tail vein of conscious mice at the times indicated in the figures were immediately centrifuged using a Beckman microcentrifuge (Beckman Instruments, UK) for 30 s at 13,000  g. The resulting plasma was then aliquoted into fresh eppendorf tubes and stored at 20 8C prior to analysis. Plasma glucose was assayed by an automated glucose oxidase procedure [30] using a Beckman Glucose Analyzer II (Beckman Instruments, Galway, Ireland). Plasma insulin was assayed by a modified dextrancoated charcoal radioimmunoassay [8].

2.4.

2.

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Statistics

Results are expressed as mean  S.E.M. Data were compared using ANOVA, followed by a Student–Newman–Keuls post hoc test. Area under the curve (AUC) analyses were calculated using the trapezoidal rule with baseline subtraction [3]. P < 0.05 was considered to be statistically significant.

3.

Results

3.1. Chronic effects of (Pro3)GIP, AM251 and combined drug administration on body weight, food intake and nonfasting concentrations of glucose and insulin Administration of AM251 alone or in combination with (Pro3)GIP significantly (P < 0.05 to <0.01) reduced energy intake throughout the treatment period (Fig. 1B). This was associated with significantly (P < 0.05 to <0.01) decreased body weights from day 12 onwards in all AM251-treated animals compared to controls. (Pro3)GIP treatment alone had no effect on energy intake, however body weights were significantly (P < 0.05) decreased from day 18 onwards compared to controls (Fig. 1A). Plasma glucose concentrations were progressively decreased in all treatment groups, this resulted in significantly (P < 0.05) lowered glucose concentrations from day 9 onwards in the

1038

peptides 29 (2008) 1036–1041

Fig. 1 – Effects of daily (Pro3)GIP, AM251 and combined drug administration on body weight (A), food intake (B) and nonfasting glucose (C) and insulin (D) in high-fat fed mice. (Pro3)GIP (25 nmol/kg body weight), AM251 (6 mg/kg body weight), a combination of both at the same respective doses or saline vehicle (control) were administered to high-fat fed mice for 22 days as indicated by the horizontal black bar. Values are mean W S.E.M. for eight mice per group. *P < 0.05 and **P < 0.01 compared to control.

(Pro3)GIP-treated mice. Similarly, treatment with AM251 alone or in combination with (Pro3)GIP resulted in significantly (P < 0.05) decreased plasma glucose levels on days 9 and 22 (Fig. 1C). These changes in glycemic status were not accompanied by elevated insulin concentrations (Fig. 1D).

3.2. Chronic effects of (Pro3)GIP, AM251 and combined drug administration on glucose tolerance Treatment with (Pro3)GIP, AM251 or combined drug administration for 22 days resulted in a significant improvement in glucose tolerance. Plasma glucose values were significantly (P < 0.05) reduced 15 min post-glucose injection in mice treated with (Pro3)GIP alone or in combination with AM251 (Fig. 2A). Moreover, all treatment groups significantly reduced the overall glycemic excursion (P < 0.05) when compared to controls (Fig. 2B). There were no observed differences in glucose-stimulated insulin release in any of the treatment groups (Fig. 2C and D).

3.3. Chronic effects of (Pro3)GIP, AM251 and combined drug administration on insulin sensitivity The hypoglycemic action of insulin was significantly (P < 0.05 to <0.01) augmented in terms of post-injection values in mice treated for 22 days with either (Pro3)GIP, AM251 or a combination of both drugs when compared to control (Fig. 3A). Furthermore, all treatment groups produced a

significant (P < 0.05) reduction in the 0–60 min overall AUC glycemic excursion (Fig. 3B).

3.4. Acute effects of AM251 on metabolic responses to glucose, insulin and feeding Acute administration of AM251 in combination with glucose had no effect on the overall glycemic (0–60 min AUC values; 537.5  74.1 mmol/(l min) vs. 633.8  91.4 mmol/(l min), respectively) or insulinotropic (0–60 min AUC values; 101.3  15.6 ng/(ml min) vs. 103.8  17.6 ng/(ml min), respectively) responses of high-fat fed mice compared to controls (data not shown). Similarly, acute administration of AM251 had no effect on the glycemic (0–105 min AUC values; 254.3  30.0 mmol/(l min) vs. 202.5  27.9 mmol/(l min), respectively) or insulin secretory (0–105 min AUC values; 36.7  4.9 ng/(ml min) vs. 40.5  4.9 ng/(ml min), respectively) responses to 15 min re-feeding compared to controls (data not shown). Food intake was marginally decreased by AM251 treatment compared to control (0.21  0.06 g/(mouse 15 min) vs. 0.15.  0.04 g/(mouse 15 min), respectively) but this failed to reach significance. Consistent with these observations, AM251 administered acutely with insulin to non-fasted high-fat fed mice had no significant effect on post-injection or overall AUC glycemic values (0–60 min AUC values; 111.0  13.1 mmol/(l min) vs. 139.5  12.8 mmol/(l min), respectively; data not shown) compared to saline-treated controls.

peptides 29 (2008) 1036–1041

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Fig. 2 – Effects of daily (Pro3)GIP, AM251 and combined drug administration on glucose tolerance and plasma insulin response to glucose in high-fat fed mice. Tests were conducted in high-fat fed mice after 22 daily injections of either (Pro3)GIP (25 nmol/kg body weight), AM251 (6 mg/kg body weight), a combination of both at the same respective doses or saline vehicle (control). Glucose (18 mmol/kg body weight) was administered by intraperitoneal injection at the time indicated by the arrow. Plasma glucose and insulin AUC values for 0–60 min post-injection are shown in the bottom panels (B and D). Values are mean W S.E.M. for eight mice per group. Mean body weights were 56.5 W 2.0, 50.3 W 1.3, 47.0 W 0.8 and 46.4 W 1.5 g for saline vehicle, (Pro3)GIP, AM251 and combined treatment groups, respectively. *P < 0.05 compared to control.

4.

Discussion

In the present study, we have evaluated the bioactivity of AM251 and possible antidiabetic potential of chronic AM251 treatment in combination with GIP receptor blockade in highfat fed mice. As expected, AM251 administration alone or in combination with (Pro3)GIP resulted in decreased food intake during the 22-day treatment period. Desensitization of the anorectic efficacy of CB1 receptor blockade has been reported in other longer term studies [4], however this was not observed in the current setting. Interestingly, CB1 receptor blockade has been shown to reduce spontaneous sucrose intake in Wistar rats but not regular chow intake, suggesting that CB1 blockade may selectively affect the intake of palatable substances [1]. While this scenario was seen to be true during the chronic treatment regime in the current setting, it does not explain the lack of a significant anorectic effect of AM251 in overnight fasted treatment naı¨ve high-fat fed mice. Nonetheless, the decrease in food intake in 22-day AM251-treated animals was associated with significantly lowered body weights. Furthermore, (Pro3)GIP treatment evoked a similar reduction in body weight without appreciable actions on food intake. This illustrates the important role of the endocannabinoids in regulation of feeding and also of GIP as a link between over nutrition and the development of obesity [25]. The beneficial effects of AM251 on body weight can easily be explained through decreased energy intake, although

blockade of CB1 receptors has been shown in other studies to effect body weight through processes that are independent of food intake [2]. Thus, more detailed studies on energy metabolism following AM251 administration are needed to clarify this matter. The actions of (Pro3)GIP are somewhat less obvious however, detailed analysis of the action of GIP on adipose tissues can go some way to resolving this issue. GIP is known to increase lipoprotein lipase activity and breakdown of circulating triglyceride, resulting in fatty acid uptake and intracellular triglyceride deposition in adipocytes [14,21]. The opposing effect of GIP receptor antagonism by (Pro3)GIP will stimulate fatty acid uptake and rapid oxidation by muscle and liver that lack GIP receptors [34], thus favoring weight reduction. This effect may be enhanced by small increases in locomotor activity observed in (Pro3)GIP-treated and GIP receptor knockout mice [13,15]. Interestingly, combined AM251 and (Pro3)GIP treatment did not evoke greater weight loss than (Pro3)GIP treatment alone. The lack of synergistic effect on body weight reduction is not easily explained, but may have been magnified if the study was carried out over a longer time course. In addition, it may be that a critical level of food intake is necessary to observe any beneficial effect of GIP receptor antagonism. Assessment of glucose tolerance at the end of the 22-day study period revealed an obvious beneficial effect of AM251 and (Pro3)GIP on glucose homeostasis. However, once again the beneficial effects of combined AM251 and (Pro3)GIP treatment were not superior to either treatment regime alone.

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peptides 29 (2008) 1036–1041

Fig. 3 – Effects of daily (Pro3)GIP, AM251 and combined drug administration on insulin sensitivity in high-fat fed mice. Tests were conducted in high-fat fed mice after 22 daily injections of either (Pro3)GIP (25 nmol/kg body weight), AM251 (6 mg/kg body weight), a combination of both at the same respective doses or saline vehicle (control). Insulin (20 U/kg body weight) was administered by intraperitoneal injection at the time indicated by the arrow. Plasma glucose AUC values for 0–60 min postinjection with identical baseline subtraction are shown in the bottom panel (B). Values are mean W S.E.M. for eight mice per group. Mean body weights were 55.9 W 2.2, 50.7 W 1.6, 46.7 W 0.9 and 46.1 W 1.6 g for saline vehicle, (Pro3)GIP, AM251 and combined treatment groups, respectively.*P < 0.05 and **P < 0.01 compared to control.

The observed benefits in glucose tolerance were not associated with significant enhancement of glucose-mediated insulin responses in any of the treatment groups. Thus, despite differing modes of action, both compounds have similar, nonadditive, effects on glucose homeostasis. (Pro3)GIP-treated animals pair-fed with the AM251 group may have revealed further insights into this paradigm, although previous studies using AM251 pair-fed mice would suggest otherwise [20]. Furthermore, it is not improbable that the decreased adiposity was responsible for the improved glucose tolerance and circulating glucose levels demonstrated in the treated

animals. Insulin sensitivity testes revealed markedly lower glucose concentrations in all treatment groups, revealing the improvement in glucose tolerance most likely stems from improved insulin sensitivity. These observations are fully consistent with previous studies demonstrating GIP and CB1 receptor inhibition are capable of countering insulin resistance in peripheral tissues [25,27]. Previous studies have shown that the beneficial metabolic effects of (Pro3)GIP are not mediated acutely, but require repeated dosing regimes before they become apparent [10–13]. This presumably reflects the need to modify metabolism and enhanced insulin sensitivity as described above and previously [25]. It is less clear whether cannabinoid CB1 receptor blockade using AM251 has reduced acute effects on glucose homeostasis. To test this, we evaluated the acute effects of AM251 on glucose tolerance, insulin sensitivity and the metabolic response to feeding. These studies were unable to demonstrate any metabolic effect of AM251, indicating that the action of CB1 receptor blockade on glucose homeostasis might be largely mediated by changes in food intake or possibly by remodeling of metabolism over time. However, it has been reported that AM251 may have potential beneficial effects on glucose uptake in muscle tissue [23], while studies in CB1 knockout mice would suggest metabolic processes other than energy intake contribute to the lean phenotype of these animals [5–7,29]. In conclusion, the present study indicates that once-daily injection of either AM251 or (Pro3)GIP represents an effective means of improving metabolic control in mice fed a high-fat diet. This is consistent with the view that GIP- and CB1receptor antagonists represent an interesting new approach to the treatment of obesity and metabolic disturbances. However the lack of synergistic effect of AM251 and (Pro3)GIP warrants further study.

Acknowledgments These studies were supported by a grant from Diabetes UK and the University of Ulster Research Strategy Funding.

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