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 mice
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Ablation of gastric inhibitory polypeptide (GIP) receptor action is reported to protect against obesity and associated metabolic abnormalities. The aim of this study was to use prediabetic ob/ob mice to examine whether 60 days of chemical GIP receptor ablation with (Pro3)GIP is able to counter the development of genetic obesity-related diabetes.
Materials and methods
Young (5–7 weeks) ob/ob mice received once daily i.p. injections of either saline vehicle or (Pro3)GIP (25 nmol kg−1 day−1) over a 60 day period. Food intake, body weight and circulating glucose and insulin were measured at frequent intervals. At 60 days, glucose tolerance, response to native GIP, postprandial responses, insulin sensitivity, HbA1c, circulating hormones and plasma lipids were assessed.
Body weight and food intake in (Pro3)GIP-treated mice did not differ from ob/ob controls. GIP receptor blockade significantly improved non-fasting glucose (p < 0.001), HbA1c (p < 0.05), glucose tolerance (p < 0.001), meal tolerance (p < 0.001) and insulin sensitivity (p < 0.05). Remarkably, (Pro3)GIP treatment prevented the age-related development of diabetes, as none of these parameters differed significantly between treated ob/ob mice and normal age-matched lean controls. Circulating levels of glucagon, corticosterone, adiponectin and total cholesterol were unchanged by (Pro3)GIP, while levels of triacylglycerol, LDL-cholesterol and resistin were decreased (p < 0.05) compared with those in control ob/ob mice. Plasma and pancreatic insulin concentrations were generally lower after (Pro3)GIP treatment than in control ob/ob mice (p < 0.01), but plasma insulin levels remained substantially raised (p < 0.001) compared with those observed in lean controls.
These data indicate that sustained GIP receptor antagonism provides an effective means of preventing the development of many of the metabolic abnormalities of obesity-driven diabetes.
KeywordsAntagonist GIP Glucose-dependent insulinotropic polypeptide Insulin resistance Obesity-related diabetes (Pro3)GIP
gastric inhibitory polypeptide
glucagon-like peptide 1
The most widely accepted role of gastric inhibitory polypeptide (GIP), also known as glucose-dependent insulinotropic polypeptide, is potentiation of glucose-induced insulin secretion from pancreatic beta cells . GIP is secreted from intestinal K cells in response to feeding and, together with glucagon-like peptide 1 (GLP-1), accounts for the full incretin effect . GIP and GLP-1 exert these effects through binding to specific beta cell receptors, activating adenylate cyclase and other signal recognition pathways, leading to insulin exocytosis . GIP and GLP-1 have also been reported to stimulate beta cell neogenesis, differentiation and proliferation whilst affording protection from cytokine attack and apoptosis [4, 5, 6]. These various attributes, plus reports of GLP-1-stimulated decreases of gastric emptying, glucagon secretion and appetite , have focused much attention on the potential of incretin hormones for the treatment of type 2 diabetes [8, 9].
Both GLP-1 and GIP are rapidly degraded in the circulation by the ubiquitous enzyme dipeptidyl-peptidase IV (DPPIV) , but problems of short half-life and pharmacokinetics have been overcome by the development of peptide mimetics possessing N-terminal modifications that make them resistant to DPPIV but able to activate incretin hormone receptors [8, 9, 11]. One such GLP-1 mimetic, exendin-4, has been approved for clinical use in the USA under the tradename Byetta (Amylin Pharmaceuticals, San Diego, CA), and a second palmitoylated derivative of GLP-1, liraglutide, is in late clinical development . Similar exploitation of GIP is at the late preclinical stage, complicated to some extent by the debate that GIP but not GLP-1 action is specifically impaired in type 2 diabetes [12, 13]. This insensitivity appears most pronounced in response to continuous infusion rather than bolus injection of native GIP , possibly implicating receptor desensitisation. However, it is now accepted that there is no specific molecular defect of the GIP receptor in established diabetes, first-degree relatives or gestational diabetes [15, 16, 17]. Recognition that GLP-1 action is also impaired, to a lesser extent than GIP action, in type 2 diabetes , suggests that the diminished insulin response is linked to generalised beta cell secretory dysfunction, presumably stemming from beta cell glucose insensitivity .
Although further work is required to determine whether DPPIV-resistant GIP molecules can overcome beta cell insensitivity and eventually make it to the clinic, possibly in combination with GLP-1 mimetics to activate both arms of the enteroinsular axis, other less well-recognised actions of GIP may afford other unexpected therapeutic opportunities . The GIP receptor is expressed in various extrapancreatic sites, including adipose tissue, bone, intestine, heart, stomach and brain . In relation to this, GIP has been shown to inhibit gastric acid secretion , attenuate glucagon-stimulated glucose production , decrease hepatic insulin extraction  stimulate glucose uptake in muscle  and increase both fatty acid synthesis and lipoprotein lipase activity in adipocytes [25, 26]. The particularly potent stimulation of GIP secretion after high-fat feeding  suggests a role for GIP in fat metabolism . Consistent with this, plasma GIP concentrations have been reported to be elevated in obesity-related type 2 diabetes  and animals with obesity-related diabetes . Experimental evidence in mice also indicates that inhibition of GIP signalling by genetic knockout of the GIP receptor prevents diet-induced obesity through the preferential use of fat as an energy substrate . Furthermore, it has been shown that short-term administration of the specific and stable GIP receptor antagonist (Pro3)GIP  can reverse many of the established metabolic abnormalities in adult ob/ob mice .
In the present study we have employed young prediabetic ob/ob mice to determine whether prolonged 60 day chemical GIP receptor ablation using daily injections of (Pro3)GIP is able to counter the development and progression of obesity-related diabetes in this commonly employed animal model. The results obtained indicate that sustained GIP receptor antagonism provides an effective means of preventing the development of many of the metabolic abnormalities of obesity-driven forms of diabetes.
Materials and methods
Animals Equal numbers of young male and female obese diabetic (ob/ob) mice derived from the colony maintained at Aston University (Birmingham, UK)  were used at 5–7 weeks of age. Animals were age-matched, divided into groups and housed individually in an air-conditioned room at 22 ± 2°C with a 12 h light:12 h dark cycle (08:00–20:00 h). Age-matched, normal, lean control mice from the same colony were used for comparative purposes as appropriate. The effects of repeated daily injection of (Pro3)GIP in these lean control mice has previously been shown to result in mild impairment of glucose homeostasis . Drinking water and a standard rodent maintenance diet (Trouw Nutrition, Cheshire, UK) were freely available. All animal experiments were carried out in accordance with the UK Animals (Scientific Procedures) Act 1986.
Synthesis, purification and characterisation of (Pro3)GIP
(Pro3)GIP was sequentially synthesised on an Applied Biosystems (Foster City, CA) automated peptide synthesiser (Model 432 A), as reported previously . (Pro3)GIP was purified by reversed-phase HPLC on a Waters Millenium 2010 chromatography system (Software version 2.1.5) and subsequently characterised using electrospray ionisation mass spectrometry (ESI-MS), as described elsewhere .
Experimental protocols for in vivo studies
Over a 60 day period, the ob/ob mice received once daily i.p. injections (17:00 h) of either saline vehicle (0.9% [w/v] NaCl) or (Pro3)GIP (25 nmol/kg body weight). This dose of (Pro3)GIP was chosen on the basis of earlier studies showing blockade of glucoregulatory effects of exogenous and endogenous GIP in ob/ob mice [35, 36]. The once daily dosing regimen has also proved effective in longer-term studies [32, 34]. Food intake and body weight were recorded daily, whereas plasma glucose and insulin concentrations were monitored at intervals of 3–7 days. Blood for HbA1c and plasma for measurement of cholesterol, triacylglycerol, glucagon, corticosterone and circulating adipokines were taken on day 60. Intraperitoneal glucose tolerance (18 mmol/kg body weight), metabolic response to native GIP (25 nmol/kg body weight) and insulin sensitivity (50 U/kg body weight) tests were performed at the end of the study period. Mice fasted for 18 h were used to examine the metabolic response to 15 min feeding on day 60. All acute experiments commenced at 10:00 h. In a separate series, pancreatic tissues were excised at the end of the 60 day treatment for measurement of insulin content following extraction with 5 ml/g of ice-cold acid ethanol (750 ml ethanol, 235 ml water, 15 ml concentrated HCl). 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, Galway, Ireland) for 30 s at 13,000×g. The resulting plasma was then aliquoted into fresh Eppendorf tubes and stored at −20°C prior to analysis. Where indicated, identical experiments were performed using age-matched, normal, lean control mice. Non-fasting plasma glucose levels in the ob/ob mice were 10.9 ± 0.8 vs 7.2 ± 0.5 mmol/l in the lean controls at the beginning of the experiment (p < 0.01).
Plasma glucose was assayed by an automated glucose oxidase procedure  using a Beckman Glucose Analyzer II (Beckman Instruments). Plasma and pancreatic insulin were assayed by a modified dextran-coated charcoal radioimmunoassay . Plasma triacylglycerol and cholesterol levels were measured using a Hitachi Automatic Analyser 912 (Boehringer Mannheim, Mannheim, Germany). HbA1c was determined using a kit purchased from Chirus (Watford, UK). Glucagon, adiponectin and resistin were measured using radioimmunoassay kits 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 the manufacturers’ instructions.
Results are expressed as means±SEM. Data were compared using ANOVA, followed by a Student–Newman–Keuls post-hoc test. AUC analyses were calculated using the trapezoidal rule with baseline subtraction . LDL-cholesterol was calculated using the Friedewald Equation . Groups of data were considered to be significantly different if the p value was less than 0.05.
Effects of (Pro3)GIP on food intake, body weight, non-fasting plasma glucose and insulin and HbA1c concentrations
Effects of (Pro3)GIP on intraperitoneal glucose tolerance and response to native GIP
Effects of (Pro3)GIP on metabolic response to feeding, insulin sensitivity and pancreatic insulin content
The plasma glucose response to 15 min feeding was significantly lowered at 15, 30, 60 and 105 min (p < 0.05 to p < 0.001) in ob/ob mice treated for 60 days with (Pro3)GIP compared with control ob/ob mice. This translated into a significantly (p < 0.001) decreased overall glycaemic excursion, despite similar food intakes (0.4–0.6 g mouse−1 15 min−1). Plasma insulin levels were not significantly different between the two groups of ob/ob mice. However, the glycaemic and insulinotropic responses of both groups were significantly different from those of normal lean mice (data not shown).
Effects of (Pro3)GIP on circulating triacylglycerol and cholesterol
Effects of (Pro3)GIP on circulating glucagon, corticosterone and adipokines
It has been speculated for decades that GIP plays a role in the aetiology of obesity . More recently it has been shown that obese diabetic (ob/ob) mice have intestinal K cell hyperplasia, markedly elevated concentrations of intestinal (2.8-fold increase) and circulating GIP (15.1-fold increase) [29, 40, 41] and display a diminished insulinotropic response to native GIP . Additionally, ob/ob mice genetically manipulated to knock out GIP receptor function displayed significant amelioration of adiposity . These key observations, together with increasing awareness of the extrapancreatic actions of GIP, such as effects on lipid metabolism, have led to speculation that GIP receptor antagonism may offer a novel and potentially useful therapeutic approach to the treatment of obesity and associated metabolic abnormalities [11, 35].
In the present study, (Pro3)GIP, a specific and stable GIP receptor antagonist , was utilised to assess whether chemical ablation of GIP signalling for 60 days in young prediabetic ob/ob mice could prevent the development of diabetes and associated metabolic features of the obesity-driven syndrome. The ob/ob mouse model represents an extreme phenotype, being noted for marked hyperphagia, gross obesity and particularly severe insulin resistance, driven by leptin deficiency . As such, the ob/ob model represents an appropriate but strong challenge to potential antihyperglycaemic agents. Consistent with other observations [30, 32, 34], GIP blockade had no effect on food intake, and mice continued to thrive throughout the study period. Obesity was already well established in the ob/ob mice, which were 5–7 weeks of age at commencement of the study, and body weights increased progressively compared with lean controls. In contrast to the results of Miyawaki et al., obtained in a study that used GIP receptor knockout mice , interference of GIP signalling by daily injections of (Pro3)GIP did not significantly arrest the development of obesity as assessed by simple body weight or body weight gain measurements. Perhaps such a comparison is unfair given the narrow window of the present treatment compared with an 8 month study in ob/ob mice with lifelong knockout of the GIP receptor. Nevertheless, (Pro3)GIP did show an obvious and progressive trend towards decreased body weights over the present 60 day study. Further research is required to clarify such an effect, possibly involving more protracted observations with larger experimental groups and exploitation of dual-energy X-ray absorptiometry (DEXA) to accurately measure body fat mass. Notably, similar (Pro3)GIP treatment of mice fed high-fat and cafeteria diets resulted in significant decreases in body weight, tissue fat deposition and adipocyte size (unpublished results). Initiation of treatment in preweanling ob/ob mice before adipocyte proliferation, or consumption of a high-fat diet as opposed to the present carbohydrate-rich diet are also potentially interesting avenues for exploration.
Despite the absence of significant effects on the progression of obesity, the early administration of (Pro3)GIP to prediabetic ob/ob mice had many notable effects on metabolic features of the obesity-related diabetes syndrome. The GIP receptor antagonist significantly improved non-fasting glucose, HbA1c, i.p. glucose tolerance, meal tolerance and insulin sensitivity in ob/ob mice. Similar actions have also been noted with 11 days of (Pro3)GIP treatment in adult ob/ob mice . However, more remarkably, GIP receptor blockade prevented the age-related development of diabetes in the present study, and none of the above mentioned parameters differed significantly between (Pro3)GIP-treated ob/ob mice and normal age-matched lean control mice. Furthermore, the present observations suggest that GIP antagonism may be particularly effective therapeutically in the early stages of diabetes.
Although (Pro3)GIP-treated ob/ob mice did not develop diabetes as judged against relevant parameters in normal lean mice, chemical GIP receptor blockade did not prevent the emergence of other characteristic features of the ob/ob syndrome. Plasma and pancreatic insulin concentrations were lower in treated than in untreated ob/ob mice, but plasma insulin concentrations were still substantially raised compared with those in lean control mice. This suggests the persistence of abnormalities in counter-regulatory mechanisms, such as those manifested by the observed elevations of glucagon and corticosterone, which were offset by moderate levels of beta cell hyperactivity. For example, although there is no doubt that insulin sensitivity was greatly improved by (Pro3)GIP administration, it is unlikely to have been totally normalised in ob/ob mice as judged by simple tests based on hypoglycaemic activity of injected insulin. Circulating adipokines were not appreciably affected by (Pro3)GIP, with only a small possibly beneficial decrease in resistin observed. This does not indicate a significant GIPergic adipokine generation in ob/ob mice and questions their recently postulated role in alleviating insulin sensitivity in GIP receptor knockout mice . The abnormal lipid profile also persisted in ob/ob mice treated with (Pro3)GIP, but there were significant improvements in triacylglycerol and LDL-cholesterol. The former is in accordance with the reported elevation of GIP concentrations in hypertriglyceridaemic subjects .
Given that GIP is classically recognised as an important insulin-releasing hormone and a major component of the enteroinsular axis , the beneficial antihyperglycaemic actions of GIP receptor blockade reported here and elsewhere [30, 32] are quite notable. In normal mice, genetic knockout or chemical antagonism of the GIP receptor clearly results in mild impairments of glucose homeostasis and insulin secretion [34, 45]. Similar effects were produced by acute as opposed to longer-term administration of (Pro3)GIP to adult ob/ob mice . Viewed in simple terms, ablation of GIP receptor function in normal mice resulted in modest detrimental deficits in insulin release, as also observed when ob/ob mice were administered (Pro3)GIP acutely. However, in situations of obesity and insulin resistance, as exemplified by ob/ob mice, longer-term GIP receptor blockade resulted in significant amelioration of diabetes and associated metabolic features [30, 32]. Thus, substantial differences exist between GIP actions in obesity as opposed to normal physiology, emphasising an important pathophysiological role of GIP. Adipocyte GIP receptors may be especially important, as GIP has been shown to promote fatty acid uptake and lipogenesis [25, 26]. Since ob/ob mice exhibit leptin deficiency and low sympathetic activity , we cannot rule out additional effects, such as increased sympathetic activity and fatty acid oxidation. However, it is important to note that similar metabolic actions of (Pro3)GIP were observed in mice fed high-fat and cafeteria diets to induce obesity and insulin resistance (unpublished results). These various observations suggest that changes in fat deposition in tissues that influence insulin sensitivity, such as liver and muscle, may be particularly important.
Although the above conclusions are necessarily based on experimental studies in animal models, there are strong parallels with recent studies in humans undergoing surgical bypass for treatment of gross obesity and associated diabetes [46, 47, 48, 49]. The similarity with our results of GIP receptor blockage in ob/ob mice suggest that GIP is key in this type of surgery and links obesity to insulin resistance and diabetes in man. Accordingly, antagonism of GIP action appears to offer considerable promise for the development of a new and effective therapeutic approach to obesity-related diabetes through alleviation of insulin resistance.
These studies were supported by University of Ulster Research Strategy Funding and Diabetes UK. The authors would also like to acknowledge C. J. Bailey for donation of original ob/+ mice breeding pairs.
Conflict of interest
N. Irwin, F. P. M. O’Harte, V. A. Gault, P. Harriott and P. R. Flatt are shareholders in Diabetica.
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