Native Glucose-dependent Insulinotropic Polypeptide Research Articles

Molecular mechanisms underlying glucose-dependent insulinotropic polypeptide secretion in human duodenal organoids.

Glucose-dependent insulinotropic polypeptide (GIP) is an incretin hormone secreted by enteroendocrine K cells in the proximal small intestine. This study aimed to explore the function of human K cells at the molecular and cellular levels. CRISPR-Cas9 homology-directed repair was used to insert transgenes encoding a yellow fluorescent protein (Venus) or an Epac-based cAMP sensor (Epac-S-H187) in the GIP locus in human duodenal-derived organoids. Fluorescently labelled K cells were purified by FACS for RNA-seq and peptidomic analysis. GIP reporter organoids were employed for GIP secretion assays, live-cell imaging of Ca2+ using Fura-2 and cAMP using Epac-S-H187, and basic electrophysiological characterisation. The G protein-coupled receptor genes GPR142 and CASR were knocked out to evaluate roles in amino acid sensing. RNA-seq of human duodenal K cells revealed enrichment of several G protein-coupled receptors involved in nutrient sensing, including FFAR1, GPBAR1, GPR119, CASR and GPR142. Glucose induced action potential firing and cytosolic Ca2+ elevation and caused a 1.8-fold increase in GIP secretion, which was inhibited by the sodium glucose co-transporter 1/2 (SGLT1/2) blocker sotagliflozin. Activation of the long-chain fatty acid receptor free fatty acid receptor 1 (FFAR1) induced a 2.7-fold increase in GIP secretion, while tryptophan and phenylalanine stimulated secretion by 2.8- and 2.1-fold, respectively. While CASR knockout blunted intracellular Ca2+ responses, a CASR/GPR142 double knockout was needed to reduce GIP secretory responses to aromatic amino acids. The newly generated human organoid K cell model enables transcriptomic and functional characterisation of nutrient-sensing pathways involved in human GIP secretion. Both calcium-sensing receptor (CASR) and G protein-coupled receptor 142 (GPR142) contribute to protein-stimulated GIP secretion. This model will be further used to identify potential targets for modulation of native GIP secretion in diabetes and obesity.

Tirzepatide Immunogenicity on Pharmacokinetics, Efficacy, and Safety: Analysis of Data From Phase 3 Studies.

Antidrug antibodies (ADA) can potentially affect drug pharmacokinetics, safety, and efficacy. This work aimed to evaluate treatment-emergent (TE) ADA in tirzepatide (TZP)-treated participants across 7 phase 3 trials and their potential effect on pharmacokinetics, efficacy, and safety. ADA were assessed at baseline and throughout the study until end point, defined as week 40 (SURPASS-1, -2, and -5) or week 52 (SURPASS-3, -4, Japan-Mono, and Japan-Combo). Samples for ADA characterization were collected at SURPASS trial sites. Participants included ADA-evaluable TZP-treated patients with type 2 diabetes (N = 5025). Interventions included TZP 5, 10, or 15 mg. ADA were detected and characterized for their ability to cross-react with native glucose-dependent insulinotropic polypeptide (nGIP) and glucagon-like peptide-1 (nGLP-1), neutralize tirzepatide activity on GIP and GLP-1 receptors, and neutralize nGIP and nGLP-1. TE ADA developed in 51.1% of tirzepatide-treated patients. Proportions were similar across dose groups. Maximum ADA titers ranged from 1:20 to 1: 81 920 among TE ADA+ patients. Neutralizing antibodies (NAb) against TZP activity on GIP and GLP-1 receptors were observed in 1.9% and 2.1% of patients, respectively. Less than 1.0% of patients had cross-reactive NAb against nGIP or nGLP-1. TE ADA status, ADA titer, and NAb status had no effect on the pharmacokinetics or efficacy of TZP. More TE ADA+ patients experienced hypersensitivity reactions or injection site reactions than TE ADA- patients. The majority of hypersensitivity and injection site reactions were nonserious and nonsevere, and most events occurred and/or resolved irrespective of TE ADA status or titer. Immunogenicity did not affect TZP pharmacokinetics or efficacy. The majority of hypersensitivity or injection site reactions experienced by TE ADA+ patients were mild to moderate in severity.

Tirzepatide: First Approval.

Tirzepatide (Mounjaro™) is a single molecule that combines dual agonism of glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) receptors. Native GIP and GLP-1 are incretin hormones that stimulate insulin secretion and decrease glucagon secretion. GIP also plays a role in nutrient and energy metabolism, while GLP-1 also delays gastric emptying, supresses appetite and improves satiety. Eli Lilly is developing tirzepatide for the treatment of type 2 diabetes mellitus (T2DM), obesity, cardiovascular disorders in T2DM, heart failure, non-alcoholic steatohepatitis, obstructive sleep apnoea and for reducing mortality/morbidity in obesity. In May 2022, tirzepatide received its first approval in the USA to improve glycaemic control in adults with T2DM, as an adjunct to diet and exercise. Tirzepatide is in phase III development for heart failure, obesity and cardiovascular disorders in T2DM, and in phase II development for non-alcoholic steatohepatitis. This article summarizes the milestones in the development of tirzepatide leading to this first approval for T2DM.

742-P: Antidrug Antibodies Do Not Impact Pharmacokinetics, Efficacy, and Safety of Tirzepatide: Analysis of Data from 7 Phase 3 Studies

Tirzepatide (TZP) is a dual glucose-dependent insulinotropic polypeptide (GIP) and glucagon like peptide (GLP) -1 receptor agonist developed for the treatment of type 2 diabetes. Patients receiving TZP may develop an immune response to the 39 amino acid synthetic peptide. This study aimed to evaluate treatment emergent (TE) anti-drug antibodies (ADA) in TZP-treated participants across 7 Phase 3 trials and its potential effect on pharmacokinetics (PK) , efficacy, and safety. A multi-tiered immunogenicity testing strategy was used to detect and characterize ADA. ADA were characterized for their ability to cross react to native GIP (nGIP) and GLP-1 (nGLP-1) , neutralize TZP activity on GIP and GLP-1 receptors, and neutralize nGIP and nGLP-1. ADA were assessed at baseline and throughout the course of the study up to week 40 (SURPASS 1, 2, and 5) or week 52 (SURPASS 3, 4, Japan-mono, and Japan-combo) . Among all ADA-evaluable patients across the Phase 3 trials (N=5025) , TE ADA developed in 51.1% of patients treated with TZP, and the proportions were similar across the 3 TZP dose groups (5, 10, and 15 mg) . At baseline, 7.0% of patients had pre-existing ADA. Maximum ADA titers ranged from 1:20 to 1: 81920 (median 1:160) among TE ADA+ patients. Neutralizing antibodies (NAb) against TZP activity on GIP and GLP-1 receptors were observed in 1.9% and 2.1% of the TE ADA+ patients, respectively. Less than 1.0% of TZP-treated TE ADA+ patients had cross-reactive NAb against nGIP or nGLP-1. TE ADA status, ADA titer, and NAb had no discernible impact on PK profile nor HbA1c effects of TZP. The percentage of TE ADA+ and TE ADA- patients with hypersensitivity reactions was similar. A higher proportion of TE ADA+ patients reported injection site reactions, of which all were nonserious and non-severe, and the vast majority occurred and/or resolved irrespective of TE ADA status or titer level.Overall, immunogenicity was not associated with any impact on TZP PK, efficacy, or safety. Disclosure B. Calderon: Employee; Eli Lilly and Company. G.R. Mullins: Employee; Eli Lilly and Company. M. Hodsdon: Employee; Eli Lilly and Company. Stock/Shareholder; Eli Lilly and Company. G. Li: Employee; Eli Lilly and Company. G. Anglin: Employee; Eli Lilly and Company. Stock/Shareholder; Eli Lilly and Company. S. Urva: Employee; Eli Lilly and Company. K. Schneck: Employee; Eli Lilly and Company. J.N. Bardos: Employee; Eli Lilly and Company. Stock/Shareholder; Eli Lilly and Company. R.F. Martins: Employee; Eli Lilly and Company. Stock/Shareholder; Eli Lilly and Company. K. Brown: Employee; Eli Lilly and Company. Funding Funded by Eli Lilly and Company.

An oral GLP-1 and GIP dual receptor agonist improves metabolic disorders in high fat-fed mice

Dual activation of the glucagon-like peptide 1 (GLP-1) receptor and the glucose-dependent insulinotropic polypeptide (GIP) receptor has potential as a novel strategy for treatment of diabesity. Here, we created a hybrid peptide which we named 19W, and show that it is more stable in presence of murine plasma than exendin-4 is. In vitro studies were performed to reveal that 19W could stimulate insulin secretion from INS-1 cells in a dose-dependent manner, just like the native peptide GIP and exendin-4 do. 19W effectively evoked dose-dependent cAMP production in cells targeting both GLP-1R and GIPR. In healthy C57BL/6J mice, the single administration of 19W significantly improved glucose tolerance. When administered in combination with sodium deoxycholate (SDC), its oral hypoglycemic activity was enhanced. Pharmacokinetics studies in Wistar rats revealed that 19W was absorbed following oral uptake, while SDC increased its bioavailability. A long-term (28 days) exposure study of twice-daily oral administration to high fat-fed (HFF) mice showed that 19W significantly reduced animal food intake, body weight, fasting blood glucose, total serum cholesterol (T-CHO), non-esterified free fatty acids (NEFA), and low-density lipoprotein cholesterol (LDL-C) levels. It also significantly improved glucose tolerance and the pancreatic β/α cell ratio, and decreased the area of liver fibrosis. These results clearly demonstrate the beneficial action of this novel oral GLP-1/GIP dual receptor agonist to reduce adiposity and hyperglycemia in diabetic mice and to ameliorate liver fibrosis associated with obesity. This dual-acting peptide can be considered a good candidate for novel oral therapy to treat obesity and diabetes.

The Role of Tirzepatide, Dual GIP and GLP-1 Receptor Agonist, in the Management of Type2 Diabetes: The SURPASS Clinical Trials.

Glucagon-like peptide 1 (GLP-1) based therapy is an established treatment option for the management of type 2 diabetes mellitus (T2DM) and is recommended early in the treatment algorithm owing to glycaemic efficacy, weight reduction and favourable cardiovascular outcomes. Glucose-dependent insulinotropic polypeptide (GIP), on the other hand, was thought to have no potential as a glucose-lowering therapy because of observations showing no insulinotropic effect from supraphysiological infusion in people with T2DM. However, emerging evidence has illustrated that co-infusion of GLP-1 and GIP has a synergetic effect, resulting in significantly increased insulin response and glucagonostatic response, compared with separate administration of each hormone. These observations have led to the development of a dual GIP/GLP-1 receptor agonist, known as a ‘twincretin’. Tirzepatide is a novel dual GIP/GLP-1 receptor agonist formulated as a synthetic peptide containing 39 amino acids, based on the native GIP sequence. Pre-clinical trials and phase 1 and 2 clinical trials indicate that tirzepatide has potent glucose lowering and weight loss with adverse effects comparable to those of established GLP-1 receptor agonists. The long-term efficacy, safety and cardiovascular outcomes of tirzepatide will be investigated in the SURPASS phase 3 clinical trial programme. In this paper, we will review the pre-clinical and phase 1 and 2 trials for tirzepatide in the management of T2DM and give an overview of the SURPASS clinical trials.

A novel thrombin-based triagonist with diabetes-protective and weight-lowering potential

AimsTo investigate the diabetes-protective effect and weight-lowering potential of a novel long-acting triagonist at three metabolically related hormone receptors including glucagon-like peptide-1 (GLP-1), glucose-dependent insulinotropic polypeptide (GIP), glucagon receptors. Main methodsTriagonist were designed in an iterative manner from native GLP-1, GIP and Glucogan. Main peptide chain (termed TG peptides) and subsequently modified LTG peptides were synthesized via solid phase synthesis. In vitro receptor activity assay was performed to screen the TG peptide with most balanced potency on all three receptors. The in vitro biological activities of modified TG peptides were further investigated by albumin-binding measurement and proteolytic cleavage test. Subsequently, oral glucose tolerance test (OGTT), pharmacokinetic test and chronic study were subjected to the acute and long-term efficacy evaluation of selected fusion peptide, LTG-6. Key findingsTG-8 exhibited equally aligned constituent efficacy and supraphysiological potency on corresponding receptor without cross-reactivity. Modified TG-8, termed LTG-6, exerted the great binding affinity for human serum albumin and the enhanced rational controlled-release of TG-8 in vitro. Further OGTT in different gene knockout mice and diabetic mice demonstrated the promising hypoglycemic and insulinotropic abilities of LTG-6. After long-term treatment for 8 weeks, LTG-6 was proved superior to co-agonists to decrease the body weight and %HbA1c, improve reverse dyslipidemia and glycemic control in the DIO models. SignificanceLTG-6, as a newly designed long-acting triagonist, holds potential to correct the obesity related metabolic disorders.

Identification of plasma binding proteins for glucose-dependent insulinotropic polypeptide.

Glucose-dependent insulinotropic polypeptide (GIP), secreted from enteroendocrine K cells, has potent insulin-releasing and extrapancreatic glucoregulatory activities. However, exogenous GIP has less potent biological effects compared with another incretin hormone, GLP-1, which limits its use for the treatment of type 2 diabetes. The fate and secretion of administered native GIP remain unclear. The aim of this study was to identify plasma binding proteins for human GIP. Fluorescent-labelled GIP was added to fresh human plasma and subjected to clear native polyacrylamide gel electrophoresis (CN-PAGE). Then fluorescent protein bands were in-gel trypsin-digested and subjected to liquid chromatography tandem-mass spectrometry (LC-MS/MS) analysis, revealing the presence of albumin, immunoglobulin G (IgG) and transferrin. In contrast to GIP, the binding of fluorescent GLP-1 and glucagon to plasma protein fractions were minimal. CN-PAGE analysis of synthetic GIP incubated with human serum albumin, purified IgG or transferrin, and subsequent western blot analysis revealed that GIP binds to each of these proteins. Taken together, these results indicate that GIP readily binds to albumin, IgG and transferrin, three plasma proteins highly abundant in the human peripheral circulation. Separation of protein complexes using CN-PAGE and the identification of in-gel digested proteins by LC-MS/MS analysis provide a promising strategy to identify plasma binding proteins for bioactive peptides.

A Dipeptidyl Peptidase-4 Inhibitor but not Incretins Suppresses Abdominal Aortic Aneurysms in Angiotensin II-Infused Apolipoprotein E-Null Mice.

The main pathophysiology of abdominal aortic aneurysm (AAA) considerably overlaps with that of atherosclerosis. We reported that incretins [glucagon-like peptide (GLP)-1 and glucose-dependent insulinotropic polypeptide (GIP)] or a dipeptidyl peptidase-4 inhibitor (DPP-4I) suppressed atherosclerosis in apolipoprotein E-null (Apoe－/－) mice. Here we investigated the effects of incretin-related agents on AAA in a mouse model. Apoe－/－ mice maintained on an atherogenic diet were subcutaneously infused with saline, Ang II (2000 ng/kg/min), Ang II, and native GLP-1 (2.16 nmol/kg/day) or Ang II and native GIP (25 nmol/kg/day) for 4 weeks. DPP-4I (MK0626, 6 mg/kg/day) was provided in the diet to the Ang II-infused mice with or without incretin receptor antagonists [(Pro3) GIP and exendin (9-39)]. AAA occurred in 70% of the animals receiving Ang II. DPP-4I reduced this rate to 40% and significantly suppressed AAA dilatation, fibrosis, and thrombosis. In contrast, incretins failed to attenuate AAA. Incretin receptor blockers did not reverse the suppressive effects of DPP-4I on AAA. In the aorta, DPP-4I significantly reduced the expression of Interleukin-1β and increased that of tissue inhibitor of metalloproteinase (TIMP)-2. In addition, DPP-4I increased the ratio of TIMP-2 to matrix metalloproteinases-9. DPP-4I, MK0626, but not native incretins has protective effects against AAA in Ang II-infused Apoe－/－ mice via suppression of inflammation, proteolysis, and fibrosis in the aortic wall.

A novel acylated form of (d-Ala2)GIP with improved antidiabetic potential, lacking effect on body fat stores

BackgroundRapid enzymatic degradation of the incretin hormone, glucose-dependent insulinotropic polypeptide (GIP), limits therapeutic use of the native peptide for diabetes. However, enzymatically stable analogues of GIP, such as (d-Ala2)GIP, have been generated, but are still susceptible to renal filtration. MethodsThe present study examines the in vitro and in vivo biological actions of a novel, acylated GIP analogue, (d-Ala2)GIP[Lys37PAL]. ResultsIn BRIN-BD11 cells, (d-Ala2)GIP[Lys37PAL] concentration-dependently stimulated (p<0.05 to p<0.001) insulin secretion at 5.6 and 16.7mM glucose. Intraperitoneal administration of (d-Ala2)GIP[Lys37PAL] to normal mice 8h prior to a glucose load significantly reduced (p<0.05) the overall glycaemic excursion compared to controls, and increased (p<0.001) the insulinotropic response compared to (d-Ala2)GIP and saline treated high fat control mice. Once daily administration of (d-Ala2)GIP[Lys37PAL] for 21days in high fat fed mice did not affect energy intake, body weight or fat deposition. However, circulating blood glucose was significantly lower (p<0.05) accompanied by increased (p<0.05) insulin concentrations by day 21. In addition, (d-Ala2)GIP[Lys37PAL] treatment significantly (p<0.01) reduced the overall glycaemic excursion and increased pancreatic insulin content (p<0.05) and the insulinotropic response (p<0.01) to an exogenous glucose challenge on day 21. Chronic treatment with (d-Ala2)GIP[Lys37PAL] did not result in resistance to the metabolic effects of a bolus injection of native GIP. Finally, insulin sensitivity was significantly improved (p<0.001) in (d-Ala2)GIP[Lys37PAL] treated mice compared to high fat controls. ConclusionsThese data confirm that (d-Ala2)GIP[Lys37PAL] is a stable, long-acting potent GIP agonist. General significance(d-Ala2)GIP[Lys37PAL] may be suitable for further evaluation and future clinical development.

Dipeptidyl peptidase-4 inhibitors and prevention of bone fractures: Effects beyond glyemic control.

Dipeptidyl peptidase-4 (DPP-4) inhibitors improve glycemic control in patients with type 2 diabetes by preventing degradation of two incretin hormones, glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP). GLP-1 and GIP are secreted from the intestine on ingestion of various nutrients, and enhance insulin secretion from pancreatic β-cells glucose-dependently1. Both incretin hormones show various biological functions in addition to their glucose-dependent insulinotropic action. Thus, DPP-4 inhibitors are expected to exert extra effects on various tissues and cell types. Among them, their effects on bones are of particular interest. A recent meta-analysis of randomized clinical trials comparing DPP-4 inhibitors with placebo or active comparator drugs in patients with type 2 diabetes suggested that treatment with DPP-4 inhibitors could be associated with a reduced risk of bone fractures2. Type 2 diabetes is associated with higher bone mineral density and, paradoxically, with increased fracture risk, presumably because of impaired bone quality that causes fragility fractures even when bone mass remains normal3. Duration of more than 10 years, presence of diabetic nephropathy, presence of diabetic neuropathy and high serum levels of pentosidine are shown to be risk factors for bone fractures3. One plausible mechanism of increased risk of bone fractures in patients with type 2 diabetes relates to chronic hyperglycemia, raising concentrations of advanced glycation end-products, such as pentosidine, that increases non-enzymatic collagen cross-linking and impairs bone quality. Furthermore, accumulating evidence shows negative impacts of antidiabetic drugs, thiazolidinediones, on bone turnover and bone fractures in patients with type 2 diabetes. However, so far, no oral antidiabetic drugs have been associated clinically with a reduction of bone fractures. Thus, the current finding on DPP-4 inhibitors by Monami et al. is highly promising despite some limitations, including short duration of the trials included, bone fractures being not principal end-points, and no discrimination between sexes and between pre- and postmenopausal women; and the Monami study provides a premise to initiate randomized, prospective, long-term clinical trials evaluating the effects of DPP-4 inhibitors on bone metabolism and bone fractures in patients with type 2 diabetes. The effects of GIP and GLP-1 on bone metabolism have been well characterized mainly in rodents (Figure 1)1. Investigations on GIP receptor-deficient mice and GIP transgenic mice showed that GIP increases bone mass by acting on osteoblasts to promote bone formation after meal ingestion, and inhibiting parathyroid hormone-induced bone resorption. Furthermore, GIP administration has been shown to attenuate ovariectomy-induced bone loss in rats. In contrast, studies on GLP-1 receptor-deficient mice showed that GLP-1 controls bone resorption, likely through a calcitonin-dependent pathway. Administration of GLP-1 receptor agonist exenatide has been shown to promote bone formation in normal and streptozotocin-induced diabetic rats, suggesting its insulin-independent action. Although these lines of evidence suggest an association of GIP and GLP-1 with bone turnover, the effects of GIP and GLP-1 on human bone turnover are largely unknown. A recent study showed that 44-week exenatide treatment did not affect bone mineral density in patients with type 2 diabetes4. As aforementioned, GLP-1 action on the bone is presumably mediated through calcitonin. A series of clinical trials on liraglutide, another GLP-1 receptor agonist, showed few changes in serum calcitonin levels in patients with type 2 diabetes, suggesting that GLP-1 might not play a role in human bone metabolism. Regarding GIP, Henriksen et al.5 previously reported that postprandial reduction of bone resorption was not mediated by GIP, but GLP-2 – another intestinal hormone cosecreted with GLP-1. However, caution should be taken when interpreting their results, as they investigated the effects of subcutaneous single injections of native GIP that should be rapidly inactivated by DPP-4 before it reaches the bones. Therefore, further investigations are definitely required to understand GIP and GLP-1 actions on bone metabolism in humans. The effects of two incretin hormones, glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like polypeptide-1 (GLP-1), on bone metabolism. GIP binds to GIP receptors expressed on osteoblasts, thereby activating new bone formation. GIP also acts on osteocrasts, presumably through osteoblasts, to suppress bone resorption. In contrast, GLP-1 stimulates calcitonin secretion from the thyroid gland, which then suppresses bone resorption by osteocrasts. Prevention of bone fractures could be the tip of the iceberg among potentially beneficial effects of DPP-4 inhibitors in patients with type 2 diabetes. It has been shown that DPP-4 inhibitors target not only two incretin hormones, GIP and GLP-1, but also other DPP-4 substrates, such as pituitary adenylate cyclase-activating peptide and stromal cell-derived factor-1α in patients with type 2 diabetes. Enhancement of these bioactive polypeptides could prevent progression of diabetic micro- and macrovascular complications independently of improvement in glycemic control. In the future, clinical trials with adequately powered, prospective, controlled relevant end-points will clarify the effects of DPP-4 inhibitors beyond glycemic control. The authors have no competing financial interests to disclose.

Conformational, receptor interaction and alanine scan studies of glucose-dependent insulinotropic polypeptide

Glucose-dependent insulinotropic polypeptide (GIP) is an insulinotropic incretin hormone that stimulates insulin secretion during a meal. GIP has glucose lowering abilities and hence is considered as a potential target molecule for type 2 diabetes therapy. In this article, we present the solution structure of GIP in membrane-mimicking environments by proton NMR spectroscopy and molecular modelling. GIP adopts an α-helical conformation between residues Phe 6–Gly 31 and Ala 13–Gln 29 for micellar and bicellar media, respectively. Previously we examined the effect of N-terminal Ala substitution in GIP, but here eight GIP analogues were synthesised by replacing individual residues within the central 8–18 region with alanine. These studies showed relatively minor changes in biological activity as assessed by insulin releasing potency. However, at higher concentration, GIP(Ala 16), and GIP(Ala 18) showed insulin secreting activity higher than the native GIP (P < 0.01 to P < 0.001) in cultured pancreatic BRIN-BD11 cells. Receptor interaction studies of the native GIP with the extracellular domain of its receptor were performed by using two different docking algorithms. At the optimised docking conformation, the complex was stabilised by the presence of hydrophobic interactions and intermolecular hydrogen bonding. Further, we have identified some potentially important additional C-terminal interactions of GIP with its N-terminal extracellular receptor domain.

Characterization and biological actions of N-terminal truncated forms of glucose-dependent insulinotropic polypeptide

The N-terminal domain of glucose-dependent insulinotropic polypeptide (GIP) plays an important role in regulating biological activity. This study examined biological properties of several N-terminal truncated forms of GIP and two novel forms with substitutions at Phe position-6 with Arg or Val. GIP(6-42), GIP(R6-42), GIP(V6-42), GIP(7-42) and GIP(9-42) stimulated cAMP production in BRIN-BD11 cells similar to native GIP, whereas responses to GIP(3-42), GIP(4-42), GIP(5-42) and GIP(8-42) were reduced ( P < 0.01 to P < 0.001). GIP-induced cyclic AMP production was significantly inhibited by GIP(3-42), GIP(4-42), GIP(5-42), GIP(6-42), GIP(R6-42), GIP(7-42) and GIP(8-42) ( P < 0.001). Compared with native GIP, in vitro insulinotropic activity of GIP(3-42), GIP(4-42), GIP(5-42), GIP(7-42) and GIP(8-42) was reduced ( P < 0.05 to P < 0.001), with GIP(4-42), GIP(5-42), GIP(7-42) and GIP(8-42) also potently inhibiting GIP-stimulated insulin secretion ( P < 0.001). In ob/ ob mice, GIP(4-42) and GIP(8-42) increased ( P < 0.05 to P < 0.01) plasma glucose concentrations compared to the glucose-lowering action of native GIP. When GIP(8-42) was co-administered with native GIP it countered the ability of the native peptide to lower plasma glucose and increase circulating insulin concentrations. These data confirm the importance of the N-terminal region of GIP in regulating bioactivity and reveal that sequential truncation of the peptide yields novel GIP receptor antagonists which may have functional significance.

Protease-Resistant Glucose-Dependent Insulinotropic Polypeptide Agonists Facilitate Hippocampal LTP and Reverse the Impairment of LTP Induced by Beta-Amyloid

Type 2 diabetes has been identified as a risk factor for Alzheimer's disease (AD). Insulin signaling is often impaired in AD, contributing to the neurodegeneration observed in AD patients. One potential strategy to overcome this impairment is to normalize insulin signaling in the brain. In the present study, we have examined the effects of an enzyme-resistant analogue of glucose-dependent insulinotropic polypeptide (GIP), N-AcGIP, on synaptic plasticity. N-AcGIP is a stable, long-acting peptide hormone that regulates glucose homeostasis and insulin release. We tested the effects of native GIP and the agonist N-AcGIP on synaptic plasticity [long-term potentiation (LTP)] in the hippocampus [15 nmol, administered intracerebroventricularly (icv)] and report for the first time that both peptides have enhancing effects on LTP. In contrast, the antagonist of GIP, Pro(3)GIP (15 nmol icv), reduced LTP. Injection of beta-amyloid(25-35) (100 nmol), a peptide that aggregates in brains of AD patients, also impaired LTP. The injection of N-AcGIP (15 nmol icv) 30 min prior to injection of amyloid(25-35) (100 nmol icv) fully reversed the impairment of LTP induced by beta-amyloid. The results demonstrate for the first time that GIP (particularly enzyme-resistant forms) not only directly modulates neurotransmitter release and LTP formation, but also protects synapses from the detrimental effects of beta-amyloid fragments on LTP formation. The use of enzyme-resistant analogues of GIP show great promise as a potential novel treatment for preventing neurodegenerative processes in AD and other related disorders.

Characterisation and biological activity of Glu 3 amino acid substituted GIP receptor antagonists

Glucose-dependent insulinotropic polypeptide (GIP) is an important gastrointestinal hormone, which regulates insulin release and glucose homeostasis, but is rapidly inactivated by enzymatic N-terminal truncation. Here we report the enzyme resistance and biological activity of several Glu 3-substituted analogues of GIP namely; (Ala 3)GIP, (Lys 3)GIP, (Phe 3)GIP, (Trp 3)GIP and (Tyr 3)GIP. Only (Lys 3)GIP demonstrated moderately enhanced resistance to DPP-IV ( p < 0.05 to p < 0.01) compared to native GIP. All analogues demonstrated a decreased potency in cAMP production (EC 50 1.47 to 11.02 nM; p < 0.01 to p < 0.001) with (Lys 3)GIP and (Phe 3)GIP significantly inhibiting GIP-stimulated cAMP production ( p < 0.05). In BRIN-BD11 cells, (Lys 3)GIP, (Phe 3)GIP, (Trp 3)GIP and (Tyr 3)GIP did not stimulate insulin secretion with both (Lys 3)GIP and (Phe 3)GIP significantly inhibiting GIP-stimulated insulin secretion ( p < 0.05). Injection of each GIP analogue together with glucose in ob/ob mice significantly increased the glycaemic excursion compared to control ( p < 0.05 to p < 0.001). This was associated with lack of significant insulin responses. (Ala 3)GIP, (Phe 3)GIP and (Tyr 3)GIP, when administered together with GIP, significantly reduced plasma insulin ( p < 0.05 to p < 0.01) and impaired the glucose-lowering ability ( p < 0.05 to p < 0.01) of the native peptide. The DPP-IV resistance and GIP antagonism observed were similar but less pronounced than (Pro 3)GIP. These data demonstrate that position 3 amino acid substitution of GIP with (Ala 3), (Phe 3), (Tyr 3) or (Pro 3) provides a new class of functional GIP receptor antagonists.

Evaluation of the antidiabetic activity of DPP IV resistant N-terminally modified versus mid-chain acylated analogues of glucose-dependent insulinotropic polypeptide

Glucose dependent insulinotropic polypeptide (GIP) is a gastrointestinal hormone with therapeutic potential for type 2 diabetes due to its insulin-releasing and antihyperglycaemic actions. However, development of GIP-based therapies is limited by N-terminal degradation by DPP IV resulting in a very short circulating half-life. Numerous GIP analogues have now been generated exhibiting DPP IV resistance and extended bioactivity profiles. In this study, we report a direct comparison of the long-term antidiabetic actions of three such GIP molecules, N-AcGIP, GIP(Lys 37PAL) and N-AcGIP(Lys 37PAL) in obese diabetic ( ob/ ob) mice. An extended duration of action of each GIP analogue was demonstrated prior to examining the effects of once daily injections (25 nmol kg −1 body weight) over a 14-day period. Administration of either N-AcGIP, GIP(Lys 37PAL) or N-AcGIP(Lys 37PAL) significantly decreased non-fasting plasma glucose and improved glucose tolerance compared to saline treated controls. All three analogues significantly enhanced glucose and nutrient-induced insulin release, and improved insulin sensitivity. The metabolic and insulin secretory responses to native GIP were also enhanced in 14-day analogue treated mice, revealing no evidence of GIP-receptor desensitization. These effects were accompanied by significantly enhanced pancreatic insulin following N-AcGIP(Lys 37PAL) and increased islet number and islet size in all three groups. Body weight, food intake and circulating glucagon were unchanged. These data demonstrate the therapeutic potential of once daily injection of enzyme resistant GIP analogues and indicate that N-AcGIP is equally as effective as related palmitate derivatised analogues of GIP.

NMR and Alanine Scan Studies of Glucose-dependent Insulinotropic Polypeptide in Water

Glucose-dependent insulinotropic polypeptide (GIP) is an incretin hormone that stimulates the secretion of insulin after ingestion of food. GIP also promotes the synthesis of fatty acids in adipose tissue. Therefore, it is not surprising that numerous literature reports have shown that GIP is linked to diabetes and obesity-related diseases. In this study, we present the solution structure of GIP in water determined by NMR spectroscopy. The calculated structure is characterized by the presence of an alpha-helical motif between residues Ser(11) and Gln(29). The helical conformation of GIP is further supported by CD spectroscopic studies. Six GIP-(1-42)Ala(1-7) analogues were synthesized by replacing individual N-terminal residues with alanine. Alanine scan studies of these N-terminal residues showed that the GIP-(1-42)Ala(6) was the only analogue to show insulin-secreting activity similar to that of the native GIP. However, when compared with glucose, its insulinotropic ability was reduced. For the first time, these NMR and modeling results contribute to the understanding of the structural requirements for the biological activity of GIP.

Biological activity and antidiabetic potential of synthetic fragment peptides of glucose-dependent insulinotropic polypeptide, GIP(1-16) and (Pro 3)GIP(1-16)

Glucose-dependent insulinotropic polypeptide (GIP) is a key physiological insulin releasing peptide and potential antidiabetic agent. The present study was undertaken in an attempt to develop small molecular weight GIP agonist and antagonist molecules. The bioactivity of two modified C-terminally truncated fragment GIP peptides, GIP(1-16) and (Pro 3)GIP(1-16), was examined in terms of insulin secretion and glucose homeostasis using BRIN-BD11 cells and type 2 diabetic mice. In vitro insulin release studies demonstrated that GIP(1-16) and (Pro 3)GIP(1-16) possessed weak GIP-receptor agonist and antagonistic properties, respectively. Intraperitoneal administration of GIP(1-16) in combination with glucose to obese diabetic ( ob/ ob) mice did not effect the glycaemic excursion and had a marginal effect on insulin release. GIP(1-16) was substantially less effective than the native GIP(1-42). (Pro 3)GIP(1-16) administration significantly curtailed ( P < 0.05) the insulinotropic and glucose lowering effects of native GIP, but was significantly less effective than (Pro 3)GIP. Based on the established concept of a therapeutic benefit of GIP receptor antagonism in obesity–diabetes, ob/ ob mice received once daily injection of (Pro 3)GIP(1-16) for 14 days. No significant effects were observed on food intake, body weight, HbA 1c, glucose tolerance, metabolic response to feeding and either insulin secretion or insulin sensitivity following prolonged (Pro 3)GIP(1-16) treatment. These data demonstrate that C-terminal truncation of GIP or (Pro 3)GIP yields small molecular weight GIP molecules with significantly reduced biological activity that precludes therapeutic utility.

Prohormone Convertase 1/3 Is Essential for Processing of the Glucose-dependent Insulinotropic Polypeptide Precursor

The physiology of the incretin hormones, glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), and their role in type 2 diabetes currently attract great interest. Recently we reported an essential role for prohormone convertase (PC) 1/3 in the cleavage of intestinal proglucagon, resulting in formation of GLP-1, as demonstrated in PC1/3-deficient mice. However, little is known about the endoproteolytic processing of the GIP precursor. This study investigates the processing of proGIP in PC1/3 and PC2 null mice and in cell lines using adenovirus-mediated overexpression. Supporting a role for PC1/3 in proGIP processing, we found co-localization of GIP and PC1/3 but not PC2 in intestinal sections by immunohistochemistry, and analysis of intestinal extracts from PC1/3-deficient animals demonstrated severely impaired processing to GIP, whereas processing to GIP was unaltered in PC2-deficient mice. Accordingly, overexpression of preproGIP in the neuroendocrine AtT-20 cell line that expresses high levels of endogenous PC1/3 and negligible levels of PC2 resulted in production of GIP. Similar results were obtained after co-expression of preproGIP and PC1/3 in GH4 cells that express no PC2 and only low levels of PC1/3. In addition, studies in GH4 cells and the alpha-TC1.9 cell line, expressing PC2 but not PC1/3, indicate that PC2 can mediate processing to GIP but also to other fragments not found in intestinal extracts. Taken together, our data indicate that PC1/3 is essential and sufficient for the production of the intestinal incretin hormone GIP, whereas PC2, although capable of cleaving proGIP, does not participate in intestinal proGIP processing and is not found in intestinal GIP-expressing cells.

Glucose-dependent insulinotropic polypeptide – beyond the enteroinsular axis?

Purpose of review: Glucose-dependent insulinotropic polypeptide (GIP) plays an important role in the enteroinsular axis, stimulating insulin secretion. In addition, it has extrapancreatic actions, which may have pathophysiological relevance. This review highlights recent findings with regard to GIP's actions both within the enteroinsular axis and beyond it, and discusses evidence for the therapeutic potential of GIP receptor agonists and antagonists for the treatment of type 2 diabetes mellitus and possibly obesity. Recent findings: GIP signalling pathways have been investigated using transgenic animal models, either lacking or overexpressing a defective GIP receptor. The dependence of early-phase insulin potentiation by GIP on KATP channel activity, and of late-phase insulin secretion on other signals, has been demonstrated. GIP receptor agonists and antagonists resistant to enzymatic degradation and with a greater potency than native GIP have recently been developed. Their activity in animal studies suggests a novel and effective treatment of type 2 diabetes. Extrapancreatic actions of GIP have received little recent attention, with notable exceptions being the investigation of aberrant GIP receptor expression in Cushing's disease, and a possible role for GIP in vascular endothelial function. Summary: The role of GIP in stimulating insulin secretion continues to be a primary focus for research, and the availability of various GIP-receptor knockout mice have helped to elucidate GIP's signalling pathways. A range of GIP receptor agonists and antagonists show promise in the treatment of type 2 diabetes, but as yet no clinical studies have been undertaken. Studies implicating GIP beyond the enteroinsular axis remain few and often negative, with the exception of effects on the vascular endothelium and the adrenal gland. © 2006 Lippincott Williams & Wilkins.