- Email: [email protected]
Comparison of the metabolic effects of GIP receptor antagonism and PYY(3-36) receptor activation in high fat fed mice
peptides 28 (2007) 2192–2198
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/peptides
Comparison of the metabolic effects of GIP receptor antagonism and PYY(3-36) receptor activation in high fat fed mice N. Irwin *, K. Hunter, P.R. Flatt School of Biomedical Sciences, University of Ulster, Coleraine, Northern Ireland BT52 1SA, UK
article info
abstract
Article history:
Glucose-dependent insulinotropic polypeptide (GIP) and peptide YY (PYY) are secreted from
Received 26 June 2007
the intestinal K- and L-cells, respectively, following a meal. Both peptides are believed to
Received in revised form
play a key role in glucose homeostasis and energy expenditure. This study investigated the
10 August 2007
effects of daily administration of the stable and specific GIP-R antagonist, (Pro3)GIP (25 nmol/
Accepted 14 August 2007
kg) and the endogenous truncated form of PYY, PYY(3-36) (50 nmol/kg), in mice fed with a
Published on line 19 August 2007
high fat diet. Daily i.p. injection of (Pro3)GIP, PYY(3-36) or combined peptide administration over 24 days significantly (P < 0.05–0.01) decreased body weight compared with saline-
Keywords:
treated controls without change in food intake. Plasma glucose levels and glucose tolerance
Glucose-dependent insulinotropic
were significantly (P < 0.05) lowered by (Pro3)GIP treatment alone, and in combination with
polypeptide (GIP)
PYY(3-36). These changes were accompanied by a slight improvement of insulin sensitivity
Peptide YY (PYY)
in all of the treatment groups. (Pro3)GIP treatment significantly reduced plasma corticos-
Glucose homeostasis
terone (P < 0.05), while combined administration with PYY(3-36) significantly lowered
High fat feeding
serum glucagon (P < 0.05). No appreciable changes were observed in either circulating or
Obesity
glucose-stimulated insulin secretion in all treatment groups. (Pro3)GIP-treated mice had significantly (P < 0.01) lowered fasting glucose levels and an improved (P < 0.05) glycemic response to feeding. These comparative data indicate that chemical ablation of GIP receptor action using (Pro3)GIP provides an especially effective means of countering obesity and related abnormalities induced by consumption of high fat energy rich diet. # 2007 Elsevier Inc. All rights reserved.
1.
Introduction
Glucose-dependent insulinotropic polypeptide (GIP) is a gut hormone secreted form the enteroendocrine K-cells that regulates post-prandial glucose homeostasis [7]. GIP, together with glucagon-like peptide-1 (GLP-1), accounts for the hormonal arm of the entero-insular axis [10]. Thus, the most widely accepted physiological role for GIP is glucosedependent stimulation of insulin secretion from pancreatic beta cells [21]. However, despite its classical role as an insulin-releasing intestinal hormone, recent data indicate
that GIP exerts effects on adipose tissue and lipid metabolism to promote fat deposition and subsequent insulin resistance [19]. Evidence is now accumulating to suggest a possible role for GIP receptor antagonists in the alleviation of obesity and insulin resistance [13,14,17,26]. This includes the fact that both normal and obese-diabetic ob/ob mice with genetic knockout of the GIP receptor are protected from obesity-diabetes [16,19]. Furthermore, studies in ob/ob mice have shown that sub-chronic daily administration of the specific and stable GIP receptor antagonist, (Pro3)GIP [12], can prevent or reverse many of the established metabolic
* Corresponding author. Tel.: +44 28 70 324313; fax: +44 28 70 324965. E-mail address: [email protected] (N. Irwin). 0196-9781/$ – see front matter # 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2007.08.008
peptides 28 (2007) 2192–2198
abnormalities associated with obesity-related diabetes [1,17]. Finally, daily (Pro3)GIP administration has recently been shown to protect against obesity, insulin resistance, glucose intolerance and associated disturbances in mice fed high fat diet [14]. Notwithstanding the clear potential of GIP receptor antagonism for alleviation of obesity and associated abnormalities [14,17], there is no evidence of reduced energy intake in animals with either genetic or chemical GIP receptor knockout [14,19]. Indeed, earlier studies have confirmed that GIP, unlike its sister incretin hormone GLP-1, lacks any effect on feeding activity [25]. Enhanced locomotor activity has recently been suggested as a possible contributor underlying weight loss in mice with ablated GIP signaling [16]. Conversely, however, overexpression of GIP in transgenic mice has been linked to enhanced locomotor activity [8], thus this scenario seems unlikely. Therefore, it may be possible to augment the beneficial effects of GIP receptor antagonism through a concurrent administration of therapy that reduces energy intake. Peptide YY (PYY) is a 36 amino acid hormone that is cosecreted with GLP-1 from the intestinal L-cells. Similar to GIP and GLP-1, endogenous concentrations of PYY are low in the fasting state and rise following feeding [1]. Consequently, PYY has been implicated in the physiology of obesity for several decades [24]. The DPP IV cleavage product of PYY, PYY(3-36), represents approximately 50% of total postprandial circulating PYY in humans [15]. Exciting findings have shown peripheral administration of this endogenous peptide form, PYY(3-36), inhibits energy intake in rodents [5] and man [2]. Recently, however, there have been a number of discrepancies among studies showing an anorectic effect of PYY(3-36), in terms of both potency and duration of action [3]. Thus, the present study was designed to evaluate the relative efficacy and combined activity of two promising new anti-obesity compounds, (Pro3)GIP and PYY(3-36). This will address the possible synergistic effects and putative mechanism of action of (Pro3)GIP and PYY(3-36) treatment in high fat fed mice. Effects on basal plasma glucose and insulin, glucose homeostasis, insulin sensitivity, metabolic response to feeding and circulating corticosterone and glucagon were examined.
2.
Materials and methods
2.1.
Animals
Male Swiss NIH mice at 6–8 weeks were obtained from Harlan UK Ltd. (Shaw’s Farm, Blackthorn, UK). Animals were agematched, 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). Animals received a high fat diet composed of 45% fat, 20% protein and 35% carbohydrate available ad libitum (26.15 kj/g; Special Diet Services) from the beginning of the experiment and drinking water was freely available. All animal experiments were carried out in accordance with the UK Animals (Scientific Procedures) Act 1986.
2.2.
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Experimental protocols for in vivo studies
Over a 24-day period, animals received once-daily intraperitoneal injections (17:00 h) of either saline vehicle (0.9% (w/v), NaCl), (Pro3)GIP (25 nmol/kg body wt; Sigma-Genosys Ltd., Cambridge, UK), PYY(3-36) (50 nmol/kg body wt; American Peptide Co., Sunnyvale, CA, USA) or a combination of both peptides at the same respective doses. These doses were chosen based on previous findings demonstrating the biological effectiveness of (Pro3)GIP and PYY(3-36) at these respective doses [3,14]. Food intake and body weight were recorded daily while non-fasting plasma glucose and insulin concentrations were monitored at 3–4 day intervals. Blood for the measurement of glucagon and corticosterone was taken on day 24. Intraperitoneal glucose tolerance (18 mmol/kg body wt) and insulin sensitivity (25 U/kg body wt) tests were performed at the end of the study period. Mice fasted for 18 h were used to examine the glycemic response to 15 min feeding of high fat diet. All acute experiments commenced at 10:00 h. 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.
2.3.
Biochemical analysis
Plasma glucose was assayed by an automated glucose oxidase procedure [22] using a Beckman Glucose Analyzer II (Beckman Instruments, Galway, Ireland). Plasma insulin was assayed by a modified dextran-coated charcoal radioimmunoassay [11]. Glucagon was measured using a radioimmunoassay kit from Linco Research (St. Charles, MI, USA). Corticosterone was measured similarly using a kit from MP Biomedicals (Heidelberg, Germany). All analyses were carried out according to instructions supplied by the various manufacturers.
2.4.
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 [4]. P < 0.05 was considered to be statistically significant.
3.
Results
3.1. Effects of (Pro3)GIP, PYY(3-36) and a combination of both peptides on body weight, food intake and non-fasting concentrations of glucose and insulin Administration of (Pro3)GIP, PYY(3-36) or a combination of both peptides had no effect on daily energy intake (Fig. 1) or 24day cumulative intake (data not shown). However, (Pro3)GIP treatment significantly (P < 0.05–0.01) decreased body weights from day 8 onwards. Similarly, body weights were significantly (P < 0.05–0.01) lowered from day 20 onwards in groups treated with PYY(3-36) alone or in combination with (Pro3)GIP (Fig. 1).
2194
peptides 28 (2007) 2192–2198
Plasma glucose concentrations were progressively reduced, resulting in significantly (P = 0.026) lowered glucose concentrations on day 18 onwards in the group treated with (Pro3)GIP. Treatment with (Pro3)GIP in combination with PYY(3-36) resulted in significantly (P = 0.034) lowered plasma glucose lower on day 18 only (Fig. 1). These changes in glycemic status were not accompanied by elevated insulin concentrations (Fig. 1). No differences in circulating plasma glucose or insulin was observed in mice treated with PYY(3-36) alone (Fig. 1).
3.2. Effects of (Pro3)GIP, PYY(3-36) and a combination of both peptides on glucose tolerance 3
Treatment with (Pro )GIP alone or in combination with PYY(336) for 24 days resulted in a significant improvement in glucose tolerance. Plasma glucose values were significantly (P = 0.041) reduced 60 min post-glucose injection in the (Pro3)GIP-treated group. Moreover, (Pro3)GIP produced a 25% reduction in the
overall glycemic excursion (P = 0.037). When, (Pro3)GIP administration was combined with PYY(3-36), a similar effect on the overall glycemic excursion was noted (P = 0.024) (Fig. 2). PYY(336) treatment alone had no significant effects on glycemic status compared to controls (Fig. 2). There were no observed differences in glucose-stimulated insulin release in any of the treatment groups (Fig. 2). High fat feeding significantly impaired the overall glycemic (P = 0.016) and elevated the overall insulinotropic (P < 0.019) responses to i.p. glucose when compared to mice fed normal chow (data not shown). Further detailed characteristics are given elsewhere [14].
3.3. Effects of (Pro3)GIP, PYY(3-36) and a combination of both peptides on insulin sensitivity The hypoglycemic action of insulin was significantly (P < 0.05– 0.01) augmented in terms of post-injection values in mice treated for 24 days with either (Pro3)GIP, PYY(3-36) or a
Fig. 1 – Effects of daily (Pro3)GIP, PYY(3-36) and combined peptide administration on food intake, body weight, %body weight and non-fasting glucose and insulin. (Pro3)GIP (25 nmol/kg body wt), PYY(3-36) (50 nmol/kg body wt), a combination of both peptides at the same respective doses or saline vehicle (control) were administered to high fat fed mice for 24 days as indicated by the horizontal black bar. Values are mean W S.E.M. for 8 mice. *P < 0.05 and **P < 0.01 compared to control.
peptides 28 (2007) 2192–2198
2195
Fig. 2 – Effects of daily (Pro3)GIP, PYY(3-36) and combined peptide administration on glucose tolerance and plasma insulin response to glucose. Tests were conducted in high fat fed mice after 24 daily injections of either (Pro3)GIP (25 nmol/ kg body wt), PYY(3-36) (50 nmol/kg body wt), a combination of both peptides at the same respective doses or saline (control). Glucose (18 mmol/kg) 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. Values were normalized for glucose and are mean W S.E.M. for 8 mice. *P < 0.05 compared to control.
combination of both peptides when compared to control (Fig. 3).
administration had no effect on plasma corticosterone when administered alone or in combination with (Pro3)GIP (Fig. 5).
3.4. Effects of (Pro3)GIP, PYY(3-36) and a combination of both peptides on glycemic response to feeding
4.
Plasma glucose response to 15 min feeding were significantly lowered (P = 0.044) at 15 min in mice treated with (Pro3)GIP for 24 days (Fig. 4), despite similar food intakes of 0.1–0.3 g/mouse/ 15 min. Furthermore, overnight fasting resulted in significantly (P = 0.006) lowered plasma glucose levels in mice treated with (Pro3)GIP for 24 days compared to controls (Fig. 4).
3.5. Effects of (Pro3)GIP, PYY(3-36) and a combination of both peptides on circulating glucagon and corticosterone Compared with controls, daily administration with (Pro3)GIP or PYY(3-36) alone did not significantly affect serum glucagon concentrations (Fig. 5). However, combined peptide administration resulted in significantly (P = 0.027) lowered serum glucagon levels compared to control mice. Daily (Pro3)GIP treatment significantly (P = 0.016) reduced plasma corticosterone concentrations in high fat fed mice, while PYY(3-36)
Discussion
There is growing evidence to suggest that gut-derived peptides play a role in modulating energy balance. A number of biological features of the major DPP IV cleavage product of peptide YY, PYY(3-36), suggest this hormone is a signal of nutritional status. These include influences on the hypothalamic circuits involved in food intake, energy expenditure and nutritional partitioning [20]. In addition, recent evidence has shown GIP to directly link overnutrition to obesity and that this hormone may efficiently promote fat storage and energy deposition [14,19]. Despite uncertainties concerning the exact role of GIP in lipid metabolism and energy balance, it is now well established that ablation of GIP signaling by genetic or chemical means can protect against obesity and insulin resistance [14,17,19]. In the present study, 6–8 week old normal mice were given ad libitum access to high fat (45%) diet for 24 days. Consistent with earlier observations, access to the high fat diet resulted in
2196
peptides 28 (2007) 2192–2198
Fig. 3 – Effects of daily (Pro3)GIP, PYY(3-36) and combined peptide administration on insulin sensitivity. Tests were conducted in high fat fed mice after 24 daily injections of either (Pro3)GIP (25 nmol/kg body wt), PYY(3-36) (50 nmol/ kg body wt), a combination of both peptides at the same respective doses or saline (control). Insulin (50 U/kg) was administered by intraperitoneal injection at the time indicated by the arrow. Values are mean W S.E.M. for 8 mice. *P < 0.05 and **P < 0.01 compared to control.
progressive body weight gain (31.7 0.8 vs. 37.6 1.5 g, P < 0.01; respectively), hyperglycaemia (7.9 0.6 vs. 11.3 0.8 mmol/l, P < 0.01; respectively) and glucose intolerance compared with age-matched controls on normal laboratory chow [14]. Administration of (Pro3)GIP, PYY(3-36) Fig. 5 – Effects of daily (Pro3)GIP, PYY(3-36) and combined peptide administration on circulating glucagon and corticosterone. Parameters were measured in high fat fed after 24 daily injections of either (Pro3)GIP (25 nmol/ kg body wt), PYY(3-36) (50 nmol/kg body wt), a combination of both peptides at the same respective doses or saline (control). Values are mean W S.E.M. for 8 mice. * P < 0.05 compared to control.
Fig. 4 – Effects of daily (Pro3)GIP, PYY(3-36) and combined peptide administration on glycemic response to feeding in 18 h fasted mice. Tests were conducted in high fat fed after 24 daily injections of either (Pro3)GIP (25 nmol/kg body wt), PYY(3-36) (50 nmol/kg body wt), a combination of both peptides at the same respective doses or saline (control). The horizontal black bar indicates the time of feeding (15 min). Values are mean W S.E.M. for 8 mice. *P < 0.05 and ** P < 0.01 compared to control.
or combined administration to mice for 24 days did not effect energy intake. This accords well with the view that GIP lacks effects on feeding activity [25] but contrasts with the reported satiety effects of PYY(3-36) [2]. Thus, despite an elevated dosing regimen in excess of 200 mg/kg body wt/day, we were unable to observe any effect of PYY(3-36) on energy intake or appetite behavior. Others have reported an anorectic effect of PYY(3-36) in rodents subjected to stress-induced fasting conditions [3]. However, several other laboratories have similarly failed to demonstrate chronic satiety effects of PYY(3-36) [3]. Interestingly, a recent report has shown that chronic intermittent administration of PYY(3-36) produces a sustained inhibitory effect of energy intake in lean and obese rats [6], hence an altered dosing regime may have produced more beneficial effects of PYY(336) in the current setting.
peptides 28 (2007) 2192–2198
The excessive body weight gain induced by high fat feeding was significantly decreased by daily (Pro3)GIP administration, reinforcing previous studies and those conducted using GIP receptor knockout mice [14,16]. Thus it appears that GIP signaling plays a critical role in energy dissipation under conditions of high fat feeding. PYY(3-36) treatment alone, and in combination with (Pro3)GIP, had a broadly similar effect on body weight change but was much less pronounced than (Pro3)GIP treatment alone. Thus, body weights remained similar to control animals in both PYY(3-36) treatment groups until day 20 of the study. Others have shown that the effects of anorectic PYY(3-36) are only observed in procedure-acclimatized rodents [5], which may partly explain the present observation. Daily administration of (Pro3)GIP alone or in combination with PYY(3-36) significantly reduced plasma glucose and improved glucose tolerance. These changes occurred without appreciable alterations of circulating insulin concentrations, unsurprising since neither PYY(3-36) nor GIP receptor antagonism using (Pro3)GIP is known to stimulate insulin release. This could also reflect a decreased stimulation of pancreatic beta-cells by lower ambient glucose concentrations. The present study did not reveal any changes in circulating glucagon following (Pro3)GIP or PYY(3-36) administration alone. However, combined peptide administration resulted in significantly lowered glucagon levels, contrasting the finding that neither peptide affects glucagon secretion under normal physiological conditions [14,23]. GIP has been shown to up-regulate circulating glucagon, but only at euglycemia [18]. This perhaps indicates a possible synergistic effect of PYY(336) and (Pro3)GIP in the pancreas. Corticosterone was decreased by GIP receptor blockade as observed previously [14], suggesting that inhibition of circulating or local production of corticosterone and a subsequent decrease of hepatic gluconeogenesis and insulin stimulated glucose uptake may be important for (Pro3)GIP action [9]. Treatment with either (Pro3)GIP or PYY(3-36) was also associated with lower glucose concentrations during insulin sensitivity tests. Similar actions have also been noted in both obese diabetic (ob/ob) mice and diet-induced obesity in mice treated with (Pro3)GIP [14,17]. Measurement of circulating resistin and adiponectin may have aided in uncovering the mechanisms behind this. However, our previous studies do not indicate a significant GIPergic adipokine effect of (Pro3)GIP in high fat fed mice [14]. Most importantly, the previously observed depletion of triglyceride deposits in adipose, liver and muscle of (Pro3)GIP-treated mice can be expected to significantly improve insulin sensitivity by promoting glucose uptake and suppressing hepatic glucose output. Taken together, these results suggest that treatment with (Pro3)GIP protects from obesity and the associated abnormalities of high fat feeding. There are very strong parallels between these effects and those from long-term studies in GIP receptor knockout mice [16,19]. The effects of PYY(3-36) were subdued in comparison, perhaps identifying the necessity for specific experimental conditions for PYY action. Nevertheless, it is clear from the present study that PYY(3-36) can have beneficial effects on body weight and associated metabolic disturbances, independent of changes in food intake. Some evidence was also obtained for a benefit
2197
of PYY(3-36) treatment in combination with (Pro3)GIP, but further studies are necessary to delineate any possible synergistic effects.
Acknowledgements These studies were supported by a grant from Diabetes UK and the University of Ulster Research Strategy Funding.
references
[1] Anini Y, Fu-Cheng X, Cuber JC, Kervran A, Chariot J, Roz C. Comparison of the postprandial release of peptide YY and proglucagon-derived peptides in the rat. Pflugers Arch 1999;438:299–306. [2] Batterham RL, Cowley MA, Small CJ, Herzog H, Cohen MA, Dakin CL, et al. Gut hormone PYY(3-36) physiologically inhibits food intake. Nature 2002;418:650–4. [3] Boggiano MM, Chandler PC, Oswald KD, Rodgers RJ, Blundell JE, Ishii Y, et al. PYY3-36 as an anti-obesity drug target. Obes Rev 2005;6:307–22. [4] Burington RS. Handbook of mathematical tables and formulae. New York: McGraw-Hill; 1973. [5] Challis BG, Pinnock SB, Coll AP, Carter RN, Dickson SL, O’Rahilly S. Acute effects of PYY3-36 on food intake and hypothalamic neuropeptide expression in the mouse. Biochem Biophys Res Commun 2003;311:915–9. [6] Chelikani PK, Haver AC, Reidelberger RD. Intermittent intraperitoneal infusion of peptide YY(3-36) reduces daily food intake and adiposity in obese rats. Am J Physiol Regul Integr Comp Physiol 2007;293:R39–46. [7] Creutzfeldt W. The entero-insular axis in type 2 diabetes— incretins as therapeutic agents. Exp Clin Endocrinol Diabetes 2001;109:S288–303. [8] Ding KH, Zhong Q, Xie D, Chen HX, Della-Fera MA, Bollag RJ, et al. Effects of glucose-dependent insulinotropic peptide on behavior. Peptides 2006;27:2750–5. [9] Donckier JE. Endocrine diseases and diabetes. In: Pickup JC, Williams G, editors. Textbook of diabetes. 3rd ed., Oxford: Blackwell Science Limited; 2003. p. 27.1–27.25. [10] Drucker DJ. The role of gut hormones in glucose homeostasis. J Clin Invest 2007;117:24–32. [11] Flatt PR, Bailey CJ. Abnormal plasma glucose and insulin responses in heterozygous lean (ob/+) mice. Diabetologia 1981;20:573–7. [12] Gault VA, O’Harte FP, Harriott P, Flatt PR. Characterization of the cellular and metabolic effects of a novel enzymeresistant antagonist of glucose-dependent insulinotropic polypeptide. Biochem Biophys Res Commun 2002;290: 1420–6. [13] Gault VA, Irwin N, Green BD, McCluskey JT, Greer B, Bailey CJ, et al. Chemical ablation of gastric inhibitory polypeptide receptor action by daily (Pro3)GIP administration improves glucose tolerance and ameliorates insulin resistance and abnormalities of islet structure in obesity-related diabetes. Diabetes 2005;54:2436–46. [14] Gault VA, McClean PL, Cassidy RS, Irwin N, Flatt PR. Chemical GIP receptor antagonism protects against obesity, insulin resistance, glucose intolerance and associated disturbances in mice fed high fat and cafeteria diets. Diabetologia 2007;50:1752–62. [15] Grandt D, Schimiczek M, Beglinger C, Layer P, Goebell H, Eysselein VE, et al. Two molecular forms of peptide YY (PYY) are abundant in human blood: characterization of a
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[17]
[18]
[19]
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radioimmunoassay recognizing PYY 1-36 and PYY 3-36. Regul Pept 1994;51:151–9. Hansotia T, Maida A, Flock G, Yamada Y, Tsukiyama K, Seino Y, et al. Extrapancreatic incretin receptors modulate glucose homeostasis, body weight, and energy expenditure. J Clin Invest 2007;117:143–52. Irwin N, McClean PL, O’Harte FPM, Gault VA, Harriott P, Flatt PR. Early administration of the glucose-dependent insulinotropic polypeptide receptor antagonist, (Pro3)GIP, prevents the development of diabetes and related metabolic abnormalities associated with genetically inherited obesity in ob/ob ice. Diabetologia 2007;50:1532–40. Meier JJ, Gallwitz B, Siepmann N, Holst JJ, Deacon CF, Schmidt WE, et al. Gastric inhibitory polypeptide (GIP) dose-dependently stimulates glucagon secretion in healthy human subjects at euglycaemia. Diabetologia 2003;46: 798–801. Miyawaki K, Yamada Y, Ban N, Ihara Y, Tsukiyama K, Zhou H. Inhibition of gastric inhibitory polypeptide signaling prevents obesity. Nat Med 2002;8:738–42.
[20] Neary NM, Small CJ, Bloom SR. Gut and mind. Gut 2003;52:918–21. [21] Pederson RA, Brown JC. The insulinotropic action of gastric inhibitory polypeptide in the perfused isolated rat pancreas. Endocrinology 1976;99:780–5. [22] Stevens JF. Determination of glucose by an automatic analyzer. Clin Chem Acta 1971;32:199–201. [23] Szecowka J, Tatemoto K, Rajamaki G, Efendic S. Effects of PYY and PP on endocrine pancreas. Acta Physiol Scand 1983;119:123–6. [24] Tatemoto K, Mutt V. Isolation of two novel candidate hormones using a chemical method for finding naturally occurring polypeptides. Nature 1980;285: 417–8. [25] Woods SC, West DB, Stein LJ, McKay LD, Lotter EC, Porte SG, et al. Peptides and the control of meal size. Diabetologia 1991;20:S305–13. [26] Yamada Y, Miyawaki K, Tsukiyama K, Harada N, Yamada C, Seino Y. Pancreatic and extrapancreatic effects of gastric inhibitory polypeptide. Diabetes 2006;55:S86–91.
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/peptides
Comparison of the metabolic effects of GIP receptor antagonism and PYY(3-36) receptor activation in high fat fed mice N. Irwin *, K. Hunter, P.R. Flatt School of Biomedical Sciences, University of Ulster, Coleraine, Northern Ireland BT52 1SA, UK
article info
abstract
Article history:
Glucose-dependent insulinotropic polypeptide (GIP) and peptide YY (PYY) are secreted from
Received 26 June 2007
the intestinal K- and L-cells, respectively, following a meal. Both peptides are believed to
Received in revised form
play a key role in glucose homeostasis and energy expenditure. This study investigated the
10 August 2007
effects of daily administration of the stable and specific GIP-R antagonist, (Pro3)GIP (25 nmol/
Accepted 14 August 2007
kg) and the endogenous truncated form of PYY, PYY(3-36) (50 nmol/kg), in mice fed with a
Published on line 19 August 2007
high fat diet. Daily i.p. injection of (Pro3)GIP, PYY(3-36) or combined peptide administration over 24 days significantly (P < 0.05–0.01) decreased body weight compared with saline-
Keywords:
treated controls without change in food intake. Plasma glucose levels and glucose tolerance
Glucose-dependent insulinotropic
were significantly (P < 0.05) lowered by (Pro3)GIP treatment alone, and in combination with
polypeptide (GIP)
PYY(3-36). These changes were accompanied by a slight improvement of insulin sensitivity
Peptide YY (PYY)
in all of the treatment groups. (Pro3)GIP treatment significantly reduced plasma corticos-
Glucose homeostasis
terone (P < 0.05), while combined administration with PYY(3-36) significantly lowered
High fat feeding
serum glucagon (P < 0.05). No appreciable changes were observed in either circulating or
Obesity
glucose-stimulated insulin secretion in all treatment groups. (Pro3)GIP-treated mice had significantly (P < 0.01) lowered fasting glucose levels and an improved (P < 0.05) glycemic response to feeding. These comparative data indicate that chemical ablation of GIP receptor action using (Pro3)GIP provides an especially effective means of countering obesity and related abnormalities induced by consumption of high fat energy rich diet. # 2007 Elsevier Inc. All rights reserved.
1.
Introduction
Glucose-dependent insulinotropic polypeptide (GIP) is a gut hormone secreted form the enteroendocrine K-cells that regulates post-prandial glucose homeostasis [7]. GIP, together with glucagon-like peptide-1 (GLP-1), accounts for the hormonal arm of the entero-insular axis [10]. Thus, the most widely accepted physiological role for GIP is glucosedependent stimulation of insulin secretion from pancreatic beta cells [21]. However, despite its classical role as an insulin-releasing intestinal hormone, recent data indicate
that GIP exerts effects on adipose tissue and lipid metabolism to promote fat deposition and subsequent insulin resistance [19]. Evidence is now accumulating to suggest a possible role for GIP receptor antagonists in the alleviation of obesity and insulin resistance [13,14,17,26]. This includes the fact that both normal and obese-diabetic ob/ob mice with genetic knockout of the GIP receptor are protected from obesity-diabetes [16,19]. Furthermore, studies in ob/ob mice have shown that sub-chronic daily administration of the specific and stable GIP receptor antagonist, (Pro3)GIP [12], can prevent or reverse many of the established metabolic
* Corresponding author. Tel.: +44 28 70 324313; fax: +44 28 70 324965. E-mail address: [email protected] (N. Irwin). 0196-9781/$ – see front matter # 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2007.08.008
peptides 28 (2007) 2192–2198
abnormalities associated with obesity-related diabetes [1,17]. Finally, daily (Pro3)GIP administration has recently been shown to protect against obesity, insulin resistance, glucose intolerance and associated disturbances in mice fed high fat diet [14]. Notwithstanding the clear potential of GIP receptor antagonism for alleviation of obesity and associated abnormalities [14,17], there is no evidence of reduced energy intake in animals with either genetic or chemical GIP receptor knockout [14,19]. Indeed, earlier studies have confirmed that GIP, unlike its sister incretin hormone GLP-1, lacks any effect on feeding activity [25]. Enhanced locomotor activity has recently been suggested as a possible contributor underlying weight loss in mice with ablated GIP signaling [16]. Conversely, however, overexpression of GIP in transgenic mice has been linked to enhanced locomotor activity [8], thus this scenario seems unlikely. Therefore, it may be possible to augment the beneficial effects of GIP receptor antagonism through a concurrent administration of therapy that reduces energy intake. Peptide YY (PYY) is a 36 amino acid hormone that is cosecreted with GLP-1 from the intestinal L-cells. Similar to GIP and GLP-1, endogenous concentrations of PYY are low in the fasting state and rise following feeding [1]. Consequently, PYY has been implicated in the physiology of obesity for several decades [24]. The DPP IV cleavage product of PYY, PYY(3-36), represents approximately 50% of total postprandial circulating PYY in humans [15]. Exciting findings have shown peripheral administration of this endogenous peptide form, PYY(3-36), inhibits energy intake in rodents [5] and man [2]. Recently, however, there have been a number of discrepancies among studies showing an anorectic effect of PYY(3-36), in terms of both potency and duration of action [3]. Thus, the present study was designed to evaluate the relative efficacy and combined activity of two promising new anti-obesity compounds, (Pro3)GIP and PYY(3-36). This will address the possible synergistic effects and putative mechanism of action of (Pro3)GIP and PYY(3-36) treatment in high fat fed mice. Effects on basal plasma glucose and insulin, glucose homeostasis, insulin sensitivity, metabolic response to feeding and circulating corticosterone and glucagon were examined.
2.
Materials and methods
2.1.
Animals
Male Swiss NIH mice at 6–8 weeks were obtained from Harlan UK Ltd. (Shaw’s Farm, Blackthorn, UK). Animals were agematched, 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). Animals received a high fat diet composed of 45% fat, 20% protein and 35% carbohydrate available ad libitum (26.15 kj/g; Special Diet Services) from the beginning of the experiment and drinking water was freely available. All animal experiments were carried out in accordance with the UK Animals (Scientific Procedures) Act 1986.
2.2.
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Experimental protocols for in vivo studies
Over a 24-day period, animals received once-daily intraperitoneal injections (17:00 h) of either saline vehicle (0.9% (w/v), NaCl), (Pro3)GIP (25 nmol/kg body wt; Sigma-Genosys Ltd., Cambridge, UK), PYY(3-36) (50 nmol/kg body wt; American Peptide Co., Sunnyvale, CA, USA) or a combination of both peptides at the same respective doses. These doses were chosen based on previous findings demonstrating the biological effectiveness of (Pro3)GIP and PYY(3-36) at these respective doses [3,14]. Food intake and body weight were recorded daily while non-fasting plasma glucose and insulin concentrations were monitored at 3–4 day intervals. Blood for the measurement of glucagon and corticosterone was taken on day 24. Intraperitoneal glucose tolerance (18 mmol/kg body wt) and insulin sensitivity (25 U/kg body wt) tests were performed at the end of the study period. Mice fasted for 18 h were used to examine the glycemic response to 15 min feeding of high fat diet. All acute experiments commenced at 10:00 h. 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.
2.3.
Biochemical analysis
Plasma glucose was assayed by an automated glucose oxidase procedure [22] using a Beckman Glucose Analyzer II (Beckman Instruments, Galway, Ireland). Plasma insulin was assayed by a modified dextran-coated charcoal radioimmunoassay [11]. Glucagon was measured using a radioimmunoassay kit from Linco Research (St. Charles, MI, USA). Corticosterone was measured similarly using a kit from MP Biomedicals (Heidelberg, Germany). All analyses were carried out according to instructions supplied by the various manufacturers.
2.4.
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 [4]. P < 0.05 was considered to be statistically significant.
3.
Results
3.1. Effects of (Pro3)GIP, PYY(3-36) and a combination of both peptides on body weight, food intake and non-fasting concentrations of glucose and insulin Administration of (Pro3)GIP, PYY(3-36) or a combination of both peptides had no effect on daily energy intake (Fig. 1) or 24day cumulative intake (data not shown). However, (Pro3)GIP treatment significantly (P < 0.05–0.01) decreased body weights from day 8 onwards. Similarly, body weights were significantly (P < 0.05–0.01) lowered from day 20 onwards in groups treated with PYY(3-36) alone or in combination with (Pro3)GIP (Fig. 1).
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Plasma glucose concentrations were progressively reduced, resulting in significantly (P = 0.026) lowered glucose concentrations on day 18 onwards in the group treated with (Pro3)GIP. Treatment with (Pro3)GIP in combination with PYY(3-36) resulted in significantly (P = 0.034) lowered plasma glucose lower on day 18 only (Fig. 1). These changes in glycemic status were not accompanied by elevated insulin concentrations (Fig. 1). No differences in circulating plasma glucose or insulin was observed in mice treated with PYY(3-36) alone (Fig. 1).
3.2. Effects of (Pro3)GIP, PYY(3-36) and a combination of both peptides on glucose tolerance 3
Treatment with (Pro )GIP alone or in combination with PYY(336) for 24 days resulted in a significant improvement in glucose tolerance. Plasma glucose values were significantly (P = 0.041) reduced 60 min post-glucose injection in the (Pro3)GIP-treated group. Moreover, (Pro3)GIP produced a 25% reduction in the
overall glycemic excursion (P = 0.037). When, (Pro3)GIP administration was combined with PYY(3-36), a similar effect on the overall glycemic excursion was noted (P = 0.024) (Fig. 2). PYY(336) treatment alone had no significant effects on glycemic status compared to controls (Fig. 2). There were no observed differences in glucose-stimulated insulin release in any of the treatment groups (Fig. 2). High fat feeding significantly impaired the overall glycemic (P = 0.016) and elevated the overall insulinotropic (P < 0.019) responses to i.p. glucose when compared to mice fed normal chow (data not shown). Further detailed characteristics are given elsewhere [14].
3.3. Effects of (Pro3)GIP, PYY(3-36) and a combination of both peptides on insulin sensitivity The hypoglycemic action of insulin was significantly (P < 0.05– 0.01) augmented in terms of post-injection values in mice treated for 24 days with either (Pro3)GIP, PYY(3-36) or a
Fig. 1 – Effects of daily (Pro3)GIP, PYY(3-36) and combined peptide administration on food intake, body weight, %body weight and non-fasting glucose and insulin. (Pro3)GIP (25 nmol/kg body wt), PYY(3-36) (50 nmol/kg body wt), a combination of both peptides at the same respective doses or saline vehicle (control) were administered to high fat fed mice for 24 days as indicated by the horizontal black bar. Values are mean W S.E.M. for 8 mice. *P < 0.05 and **P < 0.01 compared to control.
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Fig. 2 – Effects of daily (Pro3)GIP, PYY(3-36) and combined peptide administration on glucose tolerance and plasma insulin response to glucose. Tests were conducted in high fat fed mice after 24 daily injections of either (Pro3)GIP (25 nmol/ kg body wt), PYY(3-36) (50 nmol/kg body wt), a combination of both peptides at the same respective doses or saline (control). Glucose (18 mmol/kg) 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. Values were normalized for glucose and are mean W S.E.M. for 8 mice. *P < 0.05 compared to control.
combination of both peptides when compared to control (Fig. 3).
administration had no effect on plasma corticosterone when administered alone or in combination with (Pro3)GIP (Fig. 5).
3.4. Effects of (Pro3)GIP, PYY(3-36) and a combination of both peptides on glycemic response to feeding
4.
Plasma glucose response to 15 min feeding were significantly lowered (P = 0.044) at 15 min in mice treated with (Pro3)GIP for 24 days (Fig. 4), despite similar food intakes of 0.1–0.3 g/mouse/ 15 min. Furthermore, overnight fasting resulted in significantly (P = 0.006) lowered plasma glucose levels in mice treated with (Pro3)GIP for 24 days compared to controls (Fig. 4).
3.5. Effects of (Pro3)GIP, PYY(3-36) and a combination of both peptides on circulating glucagon and corticosterone Compared with controls, daily administration with (Pro3)GIP or PYY(3-36) alone did not significantly affect serum glucagon concentrations (Fig. 5). However, combined peptide administration resulted in significantly (P = 0.027) lowered serum glucagon levels compared to control mice. Daily (Pro3)GIP treatment significantly (P = 0.016) reduced plasma corticosterone concentrations in high fat fed mice, while PYY(3-36)
Discussion
There is growing evidence to suggest that gut-derived peptides play a role in modulating energy balance. A number of biological features of the major DPP IV cleavage product of peptide YY, PYY(3-36), suggest this hormone is a signal of nutritional status. These include influences on the hypothalamic circuits involved in food intake, energy expenditure and nutritional partitioning [20]. In addition, recent evidence has shown GIP to directly link overnutrition to obesity and that this hormone may efficiently promote fat storage and energy deposition [14,19]. Despite uncertainties concerning the exact role of GIP in lipid metabolism and energy balance, it is now well established that ablation of GIP signaling by genetic or chemical means can protect against obesity and insulin resistance [14,17,19]. In the present study, 6–8 week old normal mice were given ad libitum access to high fat (45%) diet for 24 days. Consistent with earlier observations, access to the high fat diet resulted in
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Fig. 3 – Effects of daily (Pro3)GIP, PYY(3-36) and combined peptide administration on insulin sensitivity. Tests were conducted in high fat fed mice after 24 daily injections of either (Pro3)GIP (25 nmol/kg body wt), PYY(3-36) (50 nmol/ kg body wt), a combination of both peptides at the same respective doses or saline (control). Insulin (50 U/kg) was administered by intraperitoneal injection at the time indicated by the arrow. Values are mean W S.E.M. for 8 mice. *P < 0.05 and **P < 0.01 compared to control.
progressive body weight gain (31.7 0.8 vs. 37.6 1.5 g, P < 0.01; respectively), hyperglycaemia (7.9 0.6 vs. 11.3 0.8 mmol/l, P < 0.01; respectively) and glucose intolerance compared with age-matched controls on normal laboratory chow [14]. Administration of (Pro3)GIP, PYY(3-36) Fig. 5 – Effects of daily (Pro3)GIP, PYY(3-36) and combined peptide administration on circulating glucagon and corticosterone. Parameters were measured in high fat fed after 24 daily injections of either (Pro3)GIP (25 nmol/ kg body wt), PYY(3-36) (50 nmol/kg body wt), a combination of both peptides at the same respective doses or saline (control). Values are mean W S.E.M. for 8 mice. * P < 0.05 compared to control.
Fig. 4 – Effects of daily (Pro3)GIP, PYY(3-36) and combined peptide administration on glycemic response to feeding in 18 h fasted mice. Tests were conducted in high fat fed after 24 daily injections of either (Pro3)GIP (25 nmol/kg body wt), PYY(3-36) (50 nmol/kg body wt), a combination of both peptides at the same respective doses or saline (control). The horizontal black bar indicates the time of feeding (15 min). Values are mean W S.E.M. for 8 mice. *P < 0.05 and ** P < 0.01 compared to control.
or combined administration to mice for 24 days did not effect energy intake. This accords well with the view that GIP lacks effects on feeding activity [25] but contrasts with the reported satiety effects of PYY(3-36) [2]. Thus, despite an elevated dosing regimen in excess of 200 mg/kg body wt/day, we were unable to observe any effect of PYY(3-36) on energy intake or appetite behavior. Others have reported an anorectic effect of PYY(3-36) in rodents subjected to stress-induced fasting conditions [3]. However, several other laboratories have similarly failed to demonstrate chronic satiety effects of PYY(3-36) [3]. Interestingly, a recent report has shown that chronic intermittent administration of PYY(3-36) produces a sustained inhibitory effect of energy intake in lean and obese rats [6], hence an altered dosing regime may have produced more beneficial effects of PYY(336) in the current setting.
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The excessive body weight gain induced by high fat feeding was significantly decreased by daily (Pro3)GIP administration, reinforcing previous studies and those conducted using GIP receptor knockout mice [14,16]. Thus it appears that GIP signaling plays a critical role in energy dissipation under conditions of high fat feeding. PYY(3-36) treatment alone, and in combination with (Pro3)GIP, had a broadly similar effect on body weight change but was much less pronounced than (Pro3)GIP treatment alone. Thus, body weights remained similar to control animals in both PYY(3-36) treatment groups until day 20 of the study. Others have shown that the effects of anorectic PYY(3-36) are only observed in procedure-acclimatized rodents [5], which may partly explain the present observation. Daily administration of (Pro3)GIP alone or in combination with PYY(3-36) significantly reduced plasma glucose and improved glucose tolerance. These changes occurred without appreciable alterations of circulating insulin concentrations, unsurprising since neither PYY(3-36) nor GIP receptor antagonism using (Pro3)GIP is known to stimulate insulin release. This could also reflect a decreased stimulation of pancreatic beta-cells by lower ambient glucose concentrations. The present study did not reveal any changes in circulating glucagon following (Pro3)GIP or PYY(3-36) administration alone. However, combined peptide administration resulted in significantly lowered glucagon levels, contrasting the finding that neither peptide affects glucagon secretion under normal physiological conditions [14,23]. GIP has been shown to up-regulate circulating glucagon, but only at euglycemia [18]. This perhaps indicates a possible synergistic effect of PYY(336) and (Pro3)GIP in the pancreas. Corticosterone was decreased by GIP receptor blockade as observed previously [14], suggesting that inhibition of circulating or local production of corticosterone and a subsequent decrease of hepatic gluconeogenesis and insulin stimulated glucose uptake may be important for (Pro3)GIP action [9]. Treatment with either (Pro3)GIP or PYY(3-36) was also associated with lower glucose concentrations during insulin sensitivity tests. Similar actions have also been noted in both obese diabetic (ob/ob) mice and diet-induced obesity in mice treated with (Pro3)GIP [14,17]. Measurement of circulating resistin and adiponectin may have aided in uncovering the mechanisms behind this. However, our previous studies do not indicate a significant GIPergic adipokine effect of (Pro3)GIP in high fat fed mice [14]. Most importantly, the previously observed depletion of triglyceride deposits in adipose, liver and muscle of (Pro3)GIP-treated mice can be expected to significantly improve insulin sensitivity by promoting glucose uptake and suppressing hepatic glucose output. Taken together, these results suggest that treatment with (Pro3)GIP protects from obesity and the associated abnormalities of high fat feeding. There are very strong parallels between these effects and those from long-term studies in GIP receptor knockout mice [16,19]. The effects of PYY(3-36) were subdued in comparison, perhaps identifying the necessity for specific experimental conditions for PYY action. Nevertheless, it is clear from the present study that PYY(3-36) can have beneficial effects on body weight and associated metabolic disturbances, independent of changes in food intake. Some evidence was also obtained for a benefit
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of PYY(3-36) treatment in combination with (Pro3)GIP, but further studies are necessary to delineate any possible synergistic effects.
Acknowledgements These studies were supported by a grant from Diabetes UK and the University of Ulster Research Strategy Funding.
references
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