Glucose-dependent Insulinotropic Polypeptide Analogues Research Articles

Altered desensitization and internalization patterns of rodent versus human glucose-dependent insulinotropic polypeptide (GIP) receptors. An important drug discovery challenge.

The gut hormone glucose-dependent insulinotropic polypeptide (GIP) signals via the GIP receptor (GIPR), resulting in postprandial potentiation of glucose-stimulated insulin secretion. The translation of results from rodent studies to human studies has been challenged by the unexpected effects of GIPR-targeting compounds. We, therefore, investigated the variation between species, focusing on GIPR desensitization and the role of the receptor C-terminus. The GIPR from humans, mice, rats, pigs, dogs and cats was studied in vitro for cognate ligand affinity, G protein activation (cAMP accumulation), recruitment of beta-arrestin and internalization. Variants of the mouse, rat and human GIPRs with swapped C-terminal tails were studied in parallel. The human GIPR is more prone to internalization than rodent GIPRs. Despite similar agonist affinities and potencies for Gαs activation, especially, the mouse GIPR shows reduced receptor desensitization, internalization and beta-arrestin recruitment. Using an enzyme-stabilized, long-acting GIP analogue, the species differences were even more pronounced. 'Tail-swapped' human, rat and mouse GIPRs were all fully functional in their Gαs coupling, and the mouse GIPR regained internalization and beta-arrestin 2 recruitment properties with the human tail. The human GIPR lost the ability to recruit beta-arrestin 2 when its own C-terminus was replaced by the rat or mouse tail. Desensitization of the human GIPR is dependent on the C-terminal tail. The species-dependent functionality of the C-terminal tail and the different species-dependent internalization patterns, especially between human and mouse GIPRs, are important factors influencing the preclinical evaluation of GIPR-targeting therapeutic compounds.

Insights into agonist-elicited activation of the human glucose-dependent insulinotropic polypeptide receptor

Glucose-dependent insulinotropic polypeptide (GIP) and its receptor (GIPR) are part of the incretin system that regulates glucose homeostasis. A series of GIPR residues putatively important for ligand binding and receptor activation were mutated and pharmacologically evaluated using GIPR selective agonists in cAMP accumulation, ERK1/2 phosphorylation (pERK1/2) and β-arrestin 2 recruitment assays. The impact of mutation on ligand efficacy was determined by operational modelling of experimental data for each mutant, with results mapped onto the full-length, active-state GIPR structure. Two interaction networks, comprising transmembrane helix (TM) 7, TM1 and TM2, and extracellular loop (ECL) 2, TM5 and ECL3 were revealed, respectively. Both networks were critical for Gαs-mediated cAMP accumulation and the recruitment of β-arrestin 2, however, cAMP response was more sensitive to alanine substitution, with most mutated residues displaying reduced signaling. Unlike the other two assays, activation of ERK1/2 was largely independent of the network involving ECL2, TM5 and ECL3, indicating that pERK1/2 is at least partially distinct from Gαs or β-arrestin pathways and this network is also crucial for potential biased agonism at GIPR. Collectively, our work advances understanding of the structure–function relationship of GIPR and provides a framework for the design and/or interpretation of GIP analogues with unique signaling profiles.

DAla2-GIP-GLU-PAL Protects Against Cognitive Deficits and Pathology in APP/PS1 Mice by Inhibiting Neuroinflammation and Upregulating cAMP/PKA/CREB Signaling Pathways.

Alzheimer's disease (AD) is a neurodegenerative disease characterized by progressive decline in cognitive function. Type 2 diabetes mellitus (T2DM) is an important risk factor for AD. Glucose-dependent insulinotropic polypeptide (GIP) has been identified to be effective in T2DM treatment and neuroprotection. The present study investigated the neuroprotective effects and possible mechanisms of DAla2GIP-Glu-PAL, a novel long-lasting GIP analogue, in APP/PS1 AD mice. Multiple behavioral tests were performed to examine the cognitive function of mice. In vivo hippocampus late-phase long-term potentiation (L-LTP) was recorded to reflect synaptic plasticity. Immunohistochemistry and immunofluorescence were used to examine the Aβ plaques and neuroinflammation in the brain. IL-1β, TNF-α, and cAMP/PKA/CREB signal molecules were also detected by ELISA or western blotting. DAla2GIP-Glu-PAL increased recognition index (RI) of APP/PS1 mice in novel object recognition test, elevated spontaneous alternation percentage of APP/PS1 mice in Y maze test, and increased target quadrant swimming time of APP/PS1 mice in Morris water maze test. DAla2GIP-Glu-PAL treatment enhanced in vivo L-LTP of APP/PS1 mice. DAla2GIP-Glu-PAL significantly reduced Aβ deposition, inhibited astrocyte and microglia proliferation, and weakened IL-1β and TNF-α secretion. DAla2GIP-Glu-PAL also upregulated cAMP/PKA/CREB signal transduction and inhibited NF-κB activation in the hippocampus of APP/PS1 mice. DAla2GIP-Glu-PAL can improve cognitive behavior, synaptic plasticity, and central pathological damage in APP/PS1 mice, which might be associated with the inhibition of neuroinflammation, as well as upregulation of cAMP-/PKA/CREB signaling pathway. This study suggests a potential benefit of DAla2GIP-Glu-PAL in the treatment of AD.

The novel dual glucose-dependent insulinotropic polypeptide and glucagon-like peptide-1 (GLP-1) receptor agonist tirzepatide transiently delays gastric emptying similarly to selective long-acting GLP-1 receptor agonists.

The effect of dual glucose‐dependent insulinotropic polypeptide (GIP) and glucagon‐like peptide‐1 (GLP‐1) receptor agonist (RA) tirzepatide on gastric emptying (GE) was compared to that of GLP‐1RAs in non‐clinical and clinical studies. GE was assessed following acute and chronic treatment with tirzepatide in diet‐induced obese mice versus semaglutide or long‐acting GIP analogue alone. Participants [with and without type 2 diabetes (T2DM)] from a phase 1, 4‐week multiple dose study received tirzepatide, dulaglutide or placebo. GE was assessed by acetaminophen absorption. In mice, tirzepatide delayed GE to a similar degree to that achieved with semaglutide; however, these acute inhibitory effects were abolished after 2 weeks of treatment. GIP analogue alone had no effect on GE or on GLP‐1's effect on GE. In participants with and without T2DM, once‐weekly tirzepatide (≥5 and ≥4.5 mg, respectively) delayed GE after a single dose. This effect diminished after multiple doses of tirzepatide or dulaglutide in healthy participants. In participants with T2DM treated with an escalation schedule of tirzepatide 5/5/10/10 or 5/5/10/15 mg, a residual GE delay was still observed after multiple doses. These data suggest that tirzepatide's activity on GE is comparable to that of selective GLP‐1RAs.

Incretin accelerates platelet-derived growth factor-BB-induced osteoblast migration via protein kinase A: The upregulation of p38 MAP kinase

Incretins, including glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), secreted from enteroendocrine cells after food ingestion, are currently recognized to regulate glucose metabolism through insulin secretion. We previously demonstrated that platelet-derived growth factor-BB (PDGF-BB) induces the migration of osteoblast-like MC3T3-E1 cells through mitogen-activated protein (MAP) kinases, including p38 MAP kinase. In the present study, we investigated whether or not incretins affect the osteoblast migration. The PDGF-BB-induced cell migration was significantly reinforced by GLP-1, GIP or cAMP analogues in MC3T3-E1 cells and normal human osteoblasts. The upregulated migration by GLP-1 or cAMP analogues was suppressed by H89, an inhibitor of protein kinase A. The amplification by GLP-1 of migration induced by PDGF-BB was almost completely reduced by SB203580, a p38 MAP kinase inhibitor in MC3T3-E1 cells and normal human osteoblasts. In addition, GIP markedly strengthened the PDGF-BB-induced phosphorylation of p38 MAP kinase. Exendin-4, a GLP-1 analogue, induced Rho A expression and its translocation from cytoplasm to plasma membranes in osteoblasts at the epiphyseal lines of developing mouse femurs in vivo. These results strongly suggest that incretins accelerates the PDGF-BB-induced migration of osteoblasts via protein kinase A, and the up-regulation of p38 MAP kinase is involved in this acceleration. Our findings may highlight the novel potential of incretins to bone physiology and therapeutic strategy against bone repair.

GIP analogues augment bone strength by modulating bone composition in diet-induced obesity in mice

Receptors to glucose-dependent insulinotropic polypeptide (GIP), have been identified on bone and GIP receptor (GIPr) knockout mice exhibit reduced bone strength and quality. Despite this, little is known on the potential beneficial bone effects of exogenous GIP on bone physiology. The aim of the present study was to assess whether stable GIP analogues were capable of ameliorating bone strength in mice with diet-induced obesity. The stable GIP analogue (D-Ala²)-GIP, and (D-Ala²)-GIP-Tag, a specific GIP analogue homing exclusively to bone, were employed. In vitro studies were used to assess effects of (D-Ala²)-GIP and (D-Ala²)-GIP-Tag on bone mineralization, lysyl oxidase activity, collagen maturity as well as osteoclast formation and activity. Subsequent in vivo studies employed obese-prediabetic Swiss NIH mice subjected to a 42-day period of daily administration of saline, (D-Ala²)-GIP or (D-Ala²)-GIP-Tag. In vitro studies confirmed that (D-Ala²)-GIP and (D-Ala²)-GIP-Tag had similar beneficial biological effects on bone cells. Administration of (D-Ala²)-GIP and (D-Ala²)-GIP-Tag resulted in lower blood glucose levels without any effects on body weight. Both GIP analogues augmented bone strength to a similar extent. Trabecular or cortical bone microarchitecture were not changed over the time course of the study. However, (D-Ala²)-GIP and (D-Ala²)-GIP-Tag augmented enzymatic collagen crosslinking as well as the heterogeneity of enzymatic collagen crosslinking, mineral-to-matrix ratio and significantly reduced the heterogeneity in mineral bone crystallite size. This study demonstrates that activation of skeletal GIPr by stable GIP analogues enhance bone strength in prediabetes and suggest that these analogues may be beneficial in the treatment of bone disease.

Efficacy of targeting bone-specific GIP receptor in ovariectomy-induced bone loss.

Glucose-dependent insulinotropic polypeptide (GIP) has been recognized in the last decade as an important contributor of bone remodelling and is necessary for optimal bone quality. However, GIP receptors are expressed in several tissues in the body and little is known about the direct vs indirect effects of GIP on bone remodelling and quality. The aims of the present study were to validate two new GIP analogues, called [d-Ala2]-GIP-Tag and [d-Ala2]-GIP1-30, which specifically target either bone or whole-body GIP receptors, respectively; and to ascertain the beneficial effects of GIP therapy on bone in a mouse model of ovariectomy-induced bone loss. Both GIP analogues exhibited similar binding capacities at the GIP receptor and intracellular responses as full-length GIP1-42. Furthermore, only [d-Ala2]-GIP-Tag, but not [d-Ala2]-GIP1-30, was undoubtedly found exclusively in the bone matrix and released at acidic pH. In ovariectomized animals, [d-Ala2]-GIP1-30 but not [d-Ala2]-GIP-Tag ameliorated bone stiffness at the same magnitude than alendronate treatment. Only [d-Ala2]-GIP1-30 treatment led to significant ameliorations in cortical microarchitecture. Although alendronate treatment increased the hardness of the bone matrix and the type B carbonate substitution in the hydroxyapatite crystals, none of the GIP analogues modified bone matrix composition. Interestingly, in ovariectomy-induced bone loss, [d-Ala2]-GIP-Tag failed to alter bone strength, microarchitecture and bone matrix composition. Overall, this study shows that the use of a GIP analogue that target whole-body GIP receptors might be useful to improve bone strength in ovariectomized animals.

Characterization of 111In-labeled Glucose-Dependent Insulinotropic Polypeptide as a Radiotracer for Neuroendocrine Tumors

Somatostatin receptor targeting is considered the standard nuclear medicine technique for visualization of neuroendocrine tumors (NET). Since not all NETs over-express somatostatin receptors, the search for novel targets, visualizing these NETs, is ongoing. Many NETs, expressing low somatostatin receptor levels, express glucose-dependent insulinotropic polypeptide (GIP) receptors (GIPR). Here, we evaluated the performance of [Lys37(DTPA)]N-acetyl-GIP1-42, a newly synthesized GIP analogue to investigate whether NET imaging via GIPR targeting is feasible. Therefore, [Lys37(DTPA)]N-acetyl-GIP1-42 was radiolabeled with 111In with specific activity up to 1.2 TBq/µmol and both in vitro and in vivo receptor targeting properties were examined. In vitro, [Lys37(111In-DTPA)]N-acetyl-GIP1-42 showed receptor-mediated binding to BHK-GIPR positive cells, NES2Y cells and isolated islets. In vivo, both NES2Y and GIPR-transfected BHK tumors were visualized on SPECT/CT. Furthermore, co-administration of an excess unlabeled GIP1-42 lowered tracer uptake from 0.7 ± 0.2%ID/g to 0.6 ± 0.01%ID/g (p = 0.78) in NES2Y tumors and significantly lowered tracer uptake from 3.3 ± 0.8 to 0.8 ± 0.2%ID/g (p = 0.0001) in GIPR-transfected BHK tumors. In conclusion, [Lys37(111In-DTPA)]N-acetyl-GIP1-42 shows receptor-mediated binding in various models. Furthermore, both GIPR-transfected BHK tumors and NES2Y tumors were visible on SPECT/CT using this tracer. Therefore, [Lys37(111In-DTPA)]N-acetyl-GIP1-42 SPECT seems promising for visualization of somatostatin receptor negative NETs.

RD Lawrence Lecture 2017 Incretins: the intelligent hormones in diabetes.

The incretin hormones glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) have attracted considerable scientific and clinical interest due largely to their insulin-releasing and glucose-lowering properties. Indeed, GLP-1-based therapies are now key treatment options for many people with diabetes worldwide. In contrast, GIP-based agents have yet to reach the clinic based primarily on the impaired insulinotropic action of GIP observed in people with diabetes. Nevertheless, GIP is a key physiological regulator of insulin secretion and stable forms of GIP show much promise in rodent models to alleviate diabetes-obesity. Recent studies suggest that GIP may have an important role to play in a combination therapeutic approach or bioengineered with other gut peptides. Moreover, recent experimental studies indicate that incretins also exert pleiotropic effects in regions of the brain associated with learning and memory, thereby supporting preclinical data demonstrating that incretin-based drugs improve cognitive function. This review article, based on the RD Lawrence Lecture presented at Diabetes UK Annual Professional Conference (2017), provides a brief overview of incretins with a major focus on GIP, the development of designer GIP analogues, and how these molecules can improve cognition. Thus, incretins can be considered as 'the intelligent hormones' and may hold the key to successfully treating the alarming rise in neurodegenerative disorders.

D-Ala2-GIP-glu-PAL is neuroprotective in a chronic Parkinson's disease mouse model and increases BNDF expression while reducing neuroinflammation and lipid peroxidation

Type 2 diabetes mellitus (T2DM) is a risk factor for Parkinson's disease (PD). Therefore, treatment to improve insulin resistance in T2DM may be useful for PD patients. Glucose dependent insulinotropic polypeptide (GIP) is a member of the incretin hormone family that can promote insulin release and improve insulin resistance. Several GIP analogues have been developed as potential treatments for T2DM. We had shown previously that D-Ala2-GIP-glu-PAL, a novel long-acting GIP analogue, can play a neuroprotective role in the PD mouse model induced by acute MPTP injection. The drug reduced damage to the dopaminergic neurons and increased CREB-mediated Bcl-2 expression to prevent apoptosis and reduced chronic inflammation in the brain. In the present study, we further tested the effects of chronic treatment by D-Ala2-GIP-glu-PAL in a chronic PD mouse model induced by MPTP (25mg/kg ip.) combination with probenecid (250mg/kg ip.) injection for 5 weeks. The results demonstrated that chronic treatment with D-Ala2-GIP-glu-PAL inhibits MPTP -induced Parkinsonism-like motor disorders in mice, and that the drug prevents dopaminergic neuronal loss in the substantia nigra pars compacta (SNpc). Moreover, D-Ala2-GIP-glu-PAL also inhibited the increased levels of expression of α-synuclein in the SNpc and striatum induced by MPTP. Furthermore, drug treatment reduced chronic neuroinflammation, oxidative stress and lipid peroxidation, and increased the expression of BDNF. These findings show that GIP signaling is neuroprotective and holds promise as a novel treatment of PD.

Glucagon-like peptide 1 and glucose-dependent insulinotropic polypeptide analogues as novel treatments for Alzheimer’s and Parkinson’s disease

Type 2 diabetes is a risk factor for developing chronic neurodegenerative disorders such as Alzheimer’s disease (AD) or Parkinson’s disease (PD). The underlying mechanism appears to be insulin desensitization in the brain. A range of glucagon-like peptide 1 (GLP-1) mimetics and glucose-dependent insulinotropic polypeptide (GIP) analogues initially designed to treat diabetes protected transgenic animals that model AD and toxin-based animal models of PD. Novel dual GLP-1/GIP analogues also show good neuroprotective effects. On the basis of these findings, first clinical trials have been conducted. In a pilot study on patients with AD, the GLP-1 analogue liraglutide showed good protective effects in 18F-fluorodeoxyglucose (18F-FDG)-PET brain imaging. It was found that the disease-related decay of brain activity had been completely stopped by the drug. In a pilot study in patients with PD, the GLP-1 mimetic exendin-4 showed good protection from motor and cognitive impairments. These results demonstrate the potential of developing disease-modifying treatments for AD and PD.

Glucose-dependent insulinotropic polypeptide (GIP) dose-dependently reduces osteoclast differentiation and resorption

A role for glucose-dependent insulinotropic polypeptide (GIP) in controlling bone resorption has been suspected. However uncertainty remains to identify whether GIP act directly on osteoclasts. The aim of the present study were (i) to identify in different osteoclast differentiation models (human peripheral blood mononuclear cells-PBMC, murine bone marrow macrophage-BMM and murine Raw 264.7 cells) whether GIP was capable of reducing osteoclast formation and resorption; (ii) ascertain whether the highly potent GIP analogue N-AcGIP was capable of inducing a response at lower concentrations and (iii) to decipher the molecular mechanisms responsible for such effects. [d-Ala2]-GIP dose-dependently reduced osteoclast formation at concentration as low as 1nM in human PBMC and 10nM in murine BMM cultures. Furthermore, [d-Ala2]-GIP also reduced the extent of osteoclast resorption at concentration as low as 1nM in human PBMC and murine BMM cultures. The mechanism of action of [d-Ala2]-GIP appeared to be mediated by reduction in intracellular calcium concentration and oscillation that subsequently inhibited calcineurin activity and NFATc1 nuclear translocation. The potency of the highly potent N-AcGIP was determined and highlighted an effect on osteoclast formation and resorption at concentration ten times lower than observed with [d-Ala2]-GIP in vitro. Furthermore, N-AcGIP was also capable of reducing the number of osteoclast in ovariectomized mice as well as the circulating level of type I collagen C-telopeptide. Pharmacological concentrations required for reducing osteoclast formation and resorption provide the impetus to design and exploit enzymatically stable GIP analogues for the treatment of bone resorption disorders in humans.

Pancreatic glucose-dependent insulinotropic polypeptide (GIP) (1–30) expression is upregulated in diabetes and PEGylated GIP(1–30) can suppress the progression of low-dose-STZ-induced hyperglycaemia in mice

Glucose-dependent insulinotropic polypeptide (GIP) is a peptide hormone released from gut K cells. While the predominant form is GIP(1-42), a shorter form, GIP(1-30), is produced by pancreatic alpha cells and promotes insulin secretion in a paracrine manner. Here, we elucidated whether GIP(1-30) expression is modulated in mouse models of diabetes. We then investigated whether PEGylated GIP(1-30) can improve islet function and morphology as well as suppress the progression to hyperglycaemia in mice treated with low-dose streptozotocin (LD-STZ). We examined pancreatic GIP immunoreactivity in rodent diabetic models. We synthesised [D-Ala(2)]GIP(1-30) and modified the C-terminus with polyethylene glycol (PEG) to produce a dipeptidyl peptidase-4 (DPP-4)-resistant long-acting GIP analogue, [D-Ala(2)]GIP(1-30)-PEG. We performed i.p.GTT and immunohistochemical analysis in non-diabetic and LD-STZ diabetic mice, with or without administration of [D-Ala(2)]GIP(1-30)-PEG. Pancreatic GIP expression was concomitantly enhanced with alpha cell expansion in rodent models of diabetes. Treatment with DPP-4 inhibitor decreased both the GIP- and glucagon-positive areas and preserved the insulin-positive area in LD-STZ diabetic mice. Body weight was not affected by [D-Ala(2)]GIP(1-30)-PEG in LD-STZ or non-diabetic mice. Treatment with GIP significantly ameliorated chronic hyperglycaemia and improved glucose excursions in LD-STZ mice. Treatment with GIP also reduced alpha cell expansion in the islets and suppressed plasma glucagon levels compared with non-treated LD-STZ mice. Additionally, [D-Ala(2)]GIP(1-30)-PEG preserved beta cell area via inhibition of apoptosis in LD-STZ mice. Our data suggest that GIP(1-30) expression is upregulated in diabetes, and PEGylated GIP(1-30) can suppress the progression to STZ-induced hyperglycaemia by inhibiting beta cell apoptosis and alpha cell expansion.

Neuroprotective effects of a GIP analogue in the MPTP Parkinson's disease mouse model

Parkinson's disease (PD) is a chronic neurodegenerative disease, and there is no cure for it at present. Recent research has indicated a link between type 2 diabetes mellitus (T2DM) and PD, which suggested that a treatment to improve insulin resistance for T2DM may be useful for PD patients. Glucose-dependent insulinotropic polypeptide (GIP) belongs to the incretin hormone family, which can promote insulin release and improve insulin resistance. Several GIP analogues have been developed as potential treatments for T2DM. In the present study, a novel long-lasting GIP analogue, D-Ala2-GIP-glu-PAL, has been tested in an acute PD mouse model induced by four 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) intraperitoneal injections. D-Ala2-GIP-glu-PAL treatment (25 nmol/kg ip.) for 7 days after MPTP treatment improved the locomotor and exploratory activity of mice, and improved bradykinesia and movement balance of mice. D-Ala2-GIP-glu-PAL treatment also restored tyrosine hydroxylase (TH) positive dopaminergic neuron numbers in the substantia nigra and TH levels in the striatum. D-Ala2-GIP-glu-PAL also reduced the chronic inflammation response as seen in astrocyte and microglia activation in the substantia nigra pars compacta (SNpc). D-Ala2-GIP-glu-PAL reversed the reduction of synapse numbers (synaptophysin levels), decreased the ratio of growth factor and apoptosis signaling molecules Bax/Bcl-2, and improved the decrease of p-CREBS133 growth factor signaling in the substantia nigra. Therefore, D-Ala2-GIP-glu-PAL promotes cell survival of dopaminergic neuron in the SNpc by activating the cAMP/PKA/CREB growth factor second messenger pathway that also inhibits apoptosis. The present results demonstrate that D-Ala2-GIP-glu-PAL shows promise as a novel treatment of PD.

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.

Neuroprotective effects of D-Ala2GIP on Alzheimer's disease biomarkers in an APP/PS1 mouse model

IntroductionType 2 diabetes mellitus has been identified as a risk factor for Alzheimer's disease (AD). An impairment of insulin signaling as well as a desensitization of its receptor has been found in AD brains. Glucose-dependent insulinotropic polypeptide (GIP) normalises insulin signaling by facilitating insulin release. GIP directly modulates neurotransmitter release, LTP formation, and protects synapses from the detrimental effects of beta-amyloid fragments on LTP formation, and cell proliferation of progenitor cells in the dentate gyrus. Here we investigate the potential therapeutic property of the new long lasting incretin hormone analogue D-Ala2GIP on key symptoms found in a mouse model of Alzheimer' disease (APPswe/PS1detaE9).MethodsD-Ala2GIP was injected for 21 days at 25 nmol/kg ip once daily in APP/PS1 male mice and wild type (WT) littermates aged 6 or 12 months of age. Amyloid plaque load, inflammation biomarkers, synaptic plasticity in the brain (LTP), and memory were measured.ResultsD-Ala2GIP improved memory in WT mice and rescued the cognitive decline of 12 months old APP/PS1 mice in two different memory tasks. Furthermore, deterioration of synaptic function in the dentate gyrus and cortex was prevented in 12 months old APP/PS1 mice. D-Ala2GIP facilitated synaptic plasticity in APP/PS1 and WT mice and reduced the number of amyloid plaques in the cortex of D-Ala2GIP injected APP/PS1 mice. The inflammatory response in microglia was also reduced.ConclusionThe results demonstrate that D-Ala2GIP has neuroprotective properties on key hallmarks found in AD. This finding shows that novel GIP analogues have the potential as a novel therapeutic for AD.

The incretin analogue D-Ala2GIP reduces plaque load, astrogliosis and oxidative stress in an APP/PS1 mouse model of Alzheimer’s disease

Type 2 diabetes mellitus has been identified as a risk factor for Alzheimer’s disease (AD). Insulin is a neuroprotective growth factor, and an impairment of insulin signalling has been found in AD brains. Glucose-dependent insulinotropic polypeptide (GIP), an incretin hormone, normalises insulin signalling and also acts as a neuroprotective growth factor. GIP plays an important role in memory formation, synaptic plasticity and cell proliferation. We have shown previously that the long-lasting incretin hormone analogue D-Ala2GIP protects memory formation and synaptic plasticity, reduces plaques, normalises the proliferation of stem cells, reduces the activation of microglia, and prevents the loss of synapses in the cortex of the APPswe/PS1deltaE9 mouse model of Alzheimer’s disease. D-Ala2GIP was injected for 35days at 25nmol/kg i.p. once daily in APP/PS1 male mice and wild-type (WT) littermates aged 6, 12 and 19months. In a follow-up study, we analysed plaque load, the activation of astrocytes as a means of chronic inflammation in the brain, and oxidative stress in the brains of these mice (8-oxoguanine levels). D-Ala2GIP reduced the amyloid plaque load in 12- and 19-month-old mice, and the inflammation response as shown in the reduction of activated astrocytes in 12- and 19-month old APP/PS1 mice. Chronic oxidative stress in the brain was reduced in 12- and 19-month-old mice as shown in the reduction of 8-oxoguanine levels in the cortex of D-Ala2GIP-injected APP/PS1 mice. The results demonstrate that D-Ala2GIP has neuroprotective properties on key markers found in Alzheimer’s disease. This finding shows that novel GIP analogues have the potential to be developed as novel therapeutics for Alzheimer’s disease.

Investigation of endothelial nitric oxide (NO) signalling in response to the incretin hormone glucose-dependent insulinotropic polypeptide (GIP)

The incretin hormone, GIP, is responsible for augmenting postprandial insulin release and regulation of β-cell mass. The advent of newly-developed GIP analogues, which have been demonstrated to restore glycaemic control in animal models of type 2 diabetes mellitus (T2DM), suggest that GIP-based therapies may now realise their long-held promise for the treatment of T2DM. Interestingly, we have recently shown that GIP may also exert vasodilatory actions which appear to be mediated via endothelium-dependent NO production. This may be particularly relevant to T2DM as over 60 % of associated mortality is attributable to cardiovascular disease related to dysregulation of endothelial NO homeostasis. An improved understanding of the mechanisms underlying GIP's recently-described vascular actions is clearly important. The aim of this study was therefore to dissect the precise mechanisms involved in GIP-mediated endothelial NO signalling.

Effects of acute and chronic administration of GIP analogues on cognition, synaptic plasticity and neurogenesis in mice

Type 2 diabetes is a risk factor for Alzheimer's disease. Insulin receptor desensitisation has been found in Alzheimer brains, which may be the underlying link. Glucose-dependent insulinotropic polypeptide (GIP), an incretin hormone, normalises insulin signalling in diabetes. GIP and the GIP receptors are widely expressed in the brain, and GIP has been shown to have growth factor and neuroprotective properties. Here we investigate the potential therapeutic properties of different doses of the protease resistant long-lasting GIP receptor agonist D-Ala2GIP and the antagonist (Pro3)GIP in C57Bl/6 mice. We found that after acute injection, D-Ala2GIP had few effects on general behaviour in the open field at any dose tested (2.5, 25, 100, or 250nmol/kg i.p.). In memory tests, no change was observed, whilst (Pro3)GIP at 25nmol/kg i.p. impaired memory formation. In a chronic study over 4weeks, mice injected with D-Ala2GIP (2.5 or 25nmol/kg i.p.) and (Pro3)GIP (25nmol/kg i.p.) learned a water maze task and object recognition task without impairment. In LTP recording in area CA1, both (Pro3)GIP as well as D-Ala2GIP enhanced LTP formation. In addition, the proliferation of neuronal progenitor cells in the dentate gyrus was increased both by D-Ala2GIP and (Pro3)GIP. The results show that the antagonist (Pro3)GIP has agonistic effects in chronic use, and both (Pro3)GIP and the agonist D-Ala2GIP are safe to use in wt mice and induces no major behavioural side effects nor impairments in learning whilst enhancing LTP and neuronal progenitor cell proliferation, which may be useful in treating neurodegenerative diseases.

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.