Effects of Hepatic Impairment on the Pharmacokinetics of the Dual GIP and GLP-1 Receptor Agonist Tirzepatide.

BACKGROUND AND OBJECTIVE: Tirzepatide, a novel, once-weekly, dual glucose-dependent insulinotropic polypeptide and glucagon-like peptide-1 receptor agonist, is approved in the US as a treatment for type 2 diabetes and is under development for long-term weight management, heart failure with preserved ejection fraction, and nonalcoholic steatohepatitis. This study evaluated the pharmacokinetics and tolerability of tirzepatide in participants with hepatic impairment (with or without type 2 diabetes) versus healthy participants with normal hepatic function. METHODS: Participants in this parallel, single-dose, open-label study were categorized by hepatic impairment defined by the baseline Child-Pugh (CP) score A (mild impairment; n = 6), B (moderate impairment; n = 6), or C (severe impairment; n = 7) or normal hepatic function (n = 13). All participants received a single subcutaneous 5-mg dose of tirzepatide. Blood samples were collected to determine tirzepatide plasma concentrations to estimate pharmacokinetic parameters. The primary pharmacokinetic parameters of area under the drug concentration-time curve from zero to infinity (AUC0-∞) and maximum observed drug concentration (Cmax) were evaluated using an analysis of covariance. The geometric least-squares means (LSM) and mean ratios for each group, between control and hepatic impairment levels, and the corresponding 90% confidence intervals (CIs) were estimated. The analysis of the time to maximum observed drug concentration was based on a nonparametric method. The relationships between the pharmacokinetic parameters and CP classification parameters (serum albumin level, total bilirubin level, and international normalized ratio) were also assessed. Adverse events were monitored to assess safety and tolerability. RESULTS: Tirzepatide exposure, based on AUC0-∞ and Cmax, was similar across the control and hepatic impairment groups. Statistical analysis showed no difference in the geometric LSM AUC0-∞ or Cmax between participants in the control group and the hepatic impairment groups, with the 90% CI for the ratios of geometric LSM spanning unity (AUC0-∞ ratio of geometric LSM vs control [90% CI 1.08 [0.879, 1.32], 0.960 [0.790, 1.17], and 0.852 [0.699, 1.04] and Cmax ratio of geometric LSM vs control [90% CI]: 0.916 [0.726, 1.16], 1.00 [0.802, 1.25], and 0.972 [0.784, 1.21] for mild, moderate and severe hepatic impairment groups, respectively). There was no change in median time to Cmax of tirzepatide across all groups (time to Cmax median difference vs control [90% CI]: 0 [- 4.00, 12.00], 0 [- 12.00, 12.00], and 0 [- 11.83, 4.17], respectively). There was no significant relationship between the exposure of tirzepatide and the CP score (p > 0.1 for AUC0-∞, Cmax, and apparent total body clearance). Similarly, there was no clinically relevant relationship between the exposure of tirzepatide and serum albumin level, total bilirubin level, or international normalized ratio. The geometric LSM half-life values were also similar across the control and hepatic impairment groups. No notable differences in safety profiles were observed between participants with hepatic impairment and healthy control participants. CONCLUSIONS: Tirzepatide pharmacokinetics was similar in participants with varying degrees of hepatic impairment compared with healthy participants. Thus, people with hepatic impairment treated with tirzepatide may not require dose adjustments. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov identifier number NCT03940742.

GIP and GLP-2 together improve bone turnover in humans supporting GIPR-GLP-2R co-agonists as future osteoporosis treatment.

The intestinal hormones glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-2 (GLP-2) are key regulators of postprandial bone turnover in humans. We hypothesized that GIP and GLP-2 co-administration would provide stronger effect on bone turnover than administration of the hormones separately, and tested this using subcutaneous injections of GIP and GLP-2 alone or in combination in humans. Guided by these findings, we designed series of GIPR-GLP-2R co-agonists as template for new osteoporosis treatment. The clinical experiment was a randomized cross-over design including 10 healthy men administered subcutaneous injections of GIP and GLP-2 alone or in combination. The GIPR-GLP-2R co-agonists were characterized in terms of binding and activation profiles on human and rodent GIP and GLP-2 receptors, and their pharmacokinetic (PK) profiles were improved by dipeptidyl peptidase-4 protection and site-directed lipidation. Co-administration of GIP and GLP-2 in humans resulted in an additive reduction in bone resorption superior to each hormone individually. The GIPR-GLP-2R co-agonists, designed by combining regions of importance for cognate receptor activation, obtained similar efficacies as the two native hormones and nanomolar potencies on both human receptors. The PK-improved co-agonists maintained receptor activity along with their prolonged half-lives. Finally, we found that the GIPR-GLP-2R co-agonists optimized toward the human receptors for bone remodeling are not feasible for use in rodent models. The successful development of potent and efficacious GIPR-GLP-2R co-agonists, combined with the improved effect on bone metabolism in humans by co-administration, support these co-agonists as a future osteoporosis treatment.

Pharmacokinetics of exogenous GIP(1-42) in C57Bl/6 mice; Extremely rapid degradation but marked variation between available assays.

Like other peptide hormones, glucose-dependent insulinotropic polypeptide (GIP) is rapidly cleared from the circulation. Dipeptidyl peptidase-4 (DPP-4) is known to be involved. Information on the overall pharmacokinetics of GIP in rodents is, however, lacking. We investigated the pharmacokinetics of exogenous GIP after intravenous, subcutaneous and intraperitoneal injection with and without DPP-4 inhibition in conscious female C57Bl/6 mice. Secondly, we compared total and intact GIP levels measured by an in-house RIA and commercially available ELISA kits to determine the suitability of these methods for in vivo and in vitro measurements. GIP half-life following intravenous injection amounted to 93 ± 2 s, which was extended to 5 ± 0.6 min by inhibition of DPP-4. Intact GIP levels following subcutaneous and intraperitoneal GIP administration were approximately 15 % of total GIP. The area under the curve of intact GIP (GIP exposure) following GIP injection was significantly increased by DPP-4 inhibition, whereas total GIP levels remained unchanged. We found significant variation between measurements of total, but not intact GIP performed with our in-house RIA and ELISAs in samples obtained after in vivo administration of GIP. Different preanalytical sample preparation (EDTA plasma, heparin plasma, assay buffer and PBS) significantly influenced results for all ELISA kits used. Thus, in experiments involving exogenous GIP(1-42) administration in mice, it is important to consider that this will result in a very low ratio of intact:total peptide but co-administration of a DPP-4 inhibitor greatly elevates this ratio. Furthermore, for comparison of GIP levels, it is essential to maintain uniformity concerning assay methodology and sample preparation.

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.

A novel GIP analogue, ZP4165, enhances glucagon-like peptide-1-induced body weight loss and improves glycaemic control in rodents.

AIM: To investigate the effects of the novel glucose-dependent insulinotropic polypeptide (GIP) analogue, ZP4165, on body weight and glycaemic control in rodents, and to investigate if ZP4165 modulates the anti-obesity and anti-hyperglycaemic effects of a glucagon-like peptide-1 (GLP-1) agonist (liraglutide). METHODS: The acute insulinotropic effect of ZP4165 was investigated in rats during an oral glucose tolerance test. The long-term effects of ZP4165 on body weight and glycaemic control, either alone or in combination with liraglutide, were assessed in diet-induced obese mice and diabetic db/db mice. RESULTS: ZP4165 showed insulinotropic action in rats. The GIP analogue did not alter the body weight of obese mice but enhanced GLP-1-induced weight loss. In diabetic mice, 4 weeks' dosing with ZP4165 reduced glycated haemoglobin levels vs vehicle by an extent similar to the GLP-1 agonist. CONCLUSIONS: ZP4165 potentiated the anti-obesity effect of a GLP-1 agonist in obese mice and improved glycaemic control in diabetic mice. These studies support further investigation of dual-incretin therapy as a more effective treatment option than mono GLP-1 medication for type 2 diabetes mellitus and obesity.

Sitagliptin decreases ventricular arrhythmias by attenuated glucose-dependent insulinotropic polypeptide (GIP)-dependent resistin signalling in infarcted rats.

Myocardial infarction (MI) was associated with insulin resistance, in which resistin acts as a critical mediator. We aimed to determine whether sitagliptin, a dipeptidyl peptidase-4 (DPP-4) inhibitor, can attenuate arrhythmias by regulating resistin-dependent nerve growth factor (NGF) expression in postinfarcted rats. Normoglycaemic male Wistar rats after ligating coronary artery were randomized to either vehicle or sitagliptin for 4 weeks starting 24 h after operation. Post-infarction was associated with increased myocardial noradrenaline [norepinephrine (NE)] levels and sympathetic hyperinnervation. Compared with vehicle, sympathetic innervation was blunted after administering sitagliptin, as assessed by immunofluorescent analysis of tyrosine hydroxylase, growth-associated factor 43 and neurofilament and western blotting and real-time quantitative RT-PCR of NGF. Arrhythmic scores in the sitagliptin-treated infarcted rats were significantly lower than those in vehicle. Furthermore, sitagliptin was associated with reduced resistin expression and increased Akt activity. Ex vivo studies showed that glucose-dependent insulinotropic polypeptide (GIP) infusion, but not glucagon-like peptide-1 (GLP-1), produced similar reduction in resistin levels to sitagliptin in postinfarcted rats. Furthermore, the attenuated effects of sitagliptin on NGF levels can be reversed by wortmannin (a phosphatidylinositol 3-kinase antagonist) and exogenous resistin infusion. Sitagliptin protects ventricular arrhythmias by attenuating sympathetic innervation in the non-diabetic infarcted rats. Sitagliptin attenuated resistin expression via the GIP-dependent pathway, which inhibited sympathetic innervation through a signalling pathway involving phosphatidylinositol 3-kinase (PI3K) and Akt protein.

Elimination and degradation of glucagon-like peptide-1 and glucose-dependent insulinotropic polypeptide in patients with end-stage renal disease.

CONTEXT: The affect of the kidneys in elimination and degradation of intact incretin hormones and their truncated metabolites is unclear. OBJECTIVE: To evaluate elimination and degradation of glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) in patients with dialysis-dependent kidney failure. SETTING AND DESIGN: Twelve non-diabetic patients treated with chronic hemodialysis and 12 control subjects were examined in a double-blind, randomized, matched observational study at the Department of Nephrology, Rigshospitalet, University of Copenhagen, Denmark. Over 4 separate study days, synthetic human GIP or GLP-1 was infused with or without concurrent inhibition of dipeptidyl peptidase 4 using sitagliptin or placebo. Plasma concentrations of glucose, insulin, glucagon, and intact and total forms of GLP-1 or GIP were measured repeatedly. Plasma half-life (T1/2), metabolic clearance rate (MCR), area under curve, and volume of distribution for intact and metabolite levels of GLP-1 and GIP were calculated. RESULTS: Fasting concentrations of intact GLP-1 and GIP were increased in dialysis patients (P < .001) whereas fasting levels of GLP-1 and GIP metabolites did not differ between groups (P > .738). MCRs of intact GLP-1 and GIP, and the GLP-1 metabolite were reduced in dialysis patients on the placebo day (P < .009), and T1/2 of intact and metabolite forms of GLP-1 and GIP were comparable between groups (P > .121). CONCLUSIONS: Unexpectedly, degradation and elimination of the intact and metabolite forms of GLP-1 and GIP seemed preserved, although reduced, in patients with dialysis-dependent kidney failure.

Glucose-dependent insulinotropic polypeptide receptors in most gastroenteropancreatic and bronchial neuroendocrine tumors.

CONTEXT: Gastrointestinal peptide hormone receptors overexpressed in neuroendocrine tumors (NET), such as somatostatin or glucagon-like peptide-1 (GLP-1) receptors, are used for in vivo tumor targeting. Unfortunately, not all NET express these receptors sufficiently. OBJECTIVE: Our aim was to evaluate in vitro the expression of another incretin receptor, glucose-dependent insulinotropic polypeptide (GIP) receptor, in human tumors and compare it with that in adjacent nonneoplastic tissues and also with somatostatin and GLP-1 receptor expression. METHODS: GIP receptor protein expression was qualitatively and quantitatively investigated in 260 human tumors and in nonneoplastic human tissues with receptor autoradiography using [(125)I]GIP(1-30). Pharmacological competition experiments and mRNA analysis were performed to provide proof of specificity. Somatostatin receptor and GLP-1 receptor autoradiography were performed in adjacent sections. RESULTS: GIP receptors are expressed in the majority of pancreatic, ileal, and bronchial NET. Importantly, most of the somatostatin receptor-negative NET and GLP-1 receptor-negative malignant insulinomas are GIP receptor positive. Conversely, the epithelial and stromal gastrointestinal tumors, including gastric, colonic, and hepatocellular carcinomas, cholangiocarcinomas, and gastrointestinal stromal tumors as well as lung adenocarcinomas are usually GIP receptor negative, except for 26% of pancreatic adenocarcinomas. Pancreatic islets, but not acini, are GIP receptor positive. The rank order of potencies for receptor binding and mRNA analysis by PCR reveal specific GIP receptors. CONCLUSIONS: The numerous GIP receptors in gastroenteropancreatic and bronchial NET represent novel universal molecular targets for clinical applications, in particular for in vivo scintigraphy and targeted radiotherapy. These results may also be the basis for multiple targeting, with concomitant use of GIP, somatostatin, and GLP-1 analogs as radiotracers.

On the role of glucose-dependent insulintropic polypeptide in postprandial metabolism in humans.

We investigated the role of glucose-dependent insulintropic polypeptide (GIP) in the regulation of gastric emptying (GE), appetite, energy intake (EI), energy expenditure (EE), plasma levels of triglycerides (TAG), and free fatty acids (FFA) in humans. First, 20 healthy males received intravenous infusion of GIP (0.8 pmol.kg(-1).min(-1)) or saline for 300 min during and after a fixed meal (protocol 1). GE was measured using paracetamol, appetite sensations using visual analog scales, EE using indirect calorimetry, and EI during a subsequent ad libitum meal (at 300 min). Next, 10 healthy males received intravenous infusions of Intralipid, glucose, or Intralipid plus glucose, with and without GIP (1.5 pmol.kg(-1).min(-1)) for 300 min (protocol 2). In protocol 1, GIP did not have any effect on GE, EI, EE, removal of TAG, or FFA and did not influence the subjective feeling of hunger, satiety, fullness or prospective food consumption compared with saline. In protocol 2, no difference was seen in the plasma TAG on Intralipid + GIP/saline and Intralipid + glucose + GIP/saline days. FFA concentrations were lower on Intralipid + glucose + GIP/saline days (P < 0.05) compared with Intralipid + GIP/saline days and on Intralipid + GIP day (P < 0.004) compared with Intralipid + saline day. Insulin increased on all GIP days compared with saline days (P < 0.05). In conclusion, while confirming its insulinotropic effects, these data suggest that GIP does not affect GE, appetite, energy intake, EE, or the clearance rate of the applied TAG formulation in humans. However, both insulin and GIP lower post-Intralipid FFA concentration, GIP probably via stimulation of insulin secretion, increasing FFA reesterification.

C-terminal mini-PEGylation of glucose-dependent insulinotropic polypeptide exhibits metabolic stability and improved glucose homeostasis in dietary-induced diabetes.

Glucose-dependent insulinotropic polypeptide has been proposed as a potential therapeutic for type 2 diabetes, however, efforts to bring forward this drug have been hindered due to its short circulating half-life. We have adopted a novel strategy to increase potency and prolong GIP action through C-terminal mini-PEGylation (GIP[mPEG]). In contrast to GIP, GIP[mPEG] was resistant to dipeptidylpeptidase-IV (DPP-IV) up to and including 24h. Both GIP[mPEG] and GIP concentration-dependently stimulated cAMP production (EC50 6.6 and 0.7 nM, respectively) and insulin secretion (p < 0.01 to p < 0.001) in pancreatic BRIN-BD11 cells. Acute injection of GIP[mPEG] together with glucose to high fat fed mice significantly lowered plasma glucose (p < 0.05) and increased plasma insulin responses (p < 0.05). Furthermore, GIP[mPEG] markedly lowered plasma glucose when administered 4-24h prior to a glucose load (p < 0.05). Daily administration of GIP[mPEG] for 20 days in high fat mice did not alter body weight, food intake or non-fasting plasma insulin, however, non-fasting plasma glucose concentrations were significantly lowered (p < 0.05). Moreover, glucose tolerance was significantly improved (p < 0.05) together with glucose-mediated plasma insulin responses (p < 0.05). Insulin sensitivity, pancreatic insulin content, triglyceride and adiponectin levels were not changed. In summary, these data demonstrate that C-terminal mini-PEGylation of GIP is a useful strategy to prolong metabolic stability and improve biological action thus representing a novel therapeutic option for type 2 diabetes.

GIP-(3-42) does not antagonize insulinotropic effects of GIP at physiological concentrations.

Glucose-dependent insulinotropic polypeptide [GIP-(1-42)] is degraded by dipeptidyl peptidase IV (DPP IV), forming GIP-(3-42). In mice, high concentrations of synthetic GIP-(3-42) may function as a GIP receptor antagonist, but it is unclear whether this occurs at physiological concentrations. In COS-7 cells transiently transfected with the human GIP receptor, GIP-(1-42) and -(3-42) bind with affinities (IC(50)) of 5.2 and 22 nM, respectively. GIP-(1-42) was a potent agonist, stimulating cAMP accumulation (EC(50), 13.5 pM); GIP-(3-42) alone had no effect. When incubated together with native GIP, GIP-(3-42) behaved as a weak antagonist (IC(50), 92 and 731 nM for inhibition of cAMP accumulation elicited by 10 pM and 1 nM native GIP, respectively). In the isolated perfused rat pancreas, GIP-(3-42) alone had no effect on insulin output and only reduced the response to GIP (1 nM) when coinfused in >50-fold molar excess (IC(50), 138 nM). The ability of GIP-(3-42) to affect the antihyperglycemic or insulinotropic actions of GIP-(1-42) was examined in chloralose-anesthetized pigs given intravenous glucose. Endogenous DPP IV activity was inhibited to reduce degradation of the infused GIP-(1-42), which was infused alone and together with GIP-(3-42), at rates sufficient to mimic postprandial concentrations of each peptide. Glucose, insulin, and glucagon responses were identical irrespective of whether GIP-(1-42) was infused alone or together with GIP-(3-42). We conclude that, although GIP-(3-42) can weakly antagonize cAMP accumulation and insulin output in vitro, it does not behave as a physiological antagonist in vivo.

Effect of whey on blood glucose and insulin responses to composite breakfast and lunch meals in type 2 diabetic subjects.

BACKGROUND: Whey proteins have insulinotropic effects and reduce the postprandial glycemia in healthy subjects. The mechanism is not known, but insulinogenic amino acids and the incretin hormones seem to be involved. OBJECTIVE: The aim was to evaluate whether supplementation of meals with a high glycemic index (GI) with whey proteins may increase insulin secretion and improve blood glucose control in type 2 diabetic subjects. DESIGN: Fourteen diet-treated subjects with type 2 diabetes were served a high-GI breakfast (white bread) and subsequent high-GI lunch (mashed potatoes with meatballs). The breakfast and lunch meals were supplemented with whey on one day; whey was exchanged for lean ham and lactose on another day. Venous blood samples were drawn before and during 4 h after breakfast and 3 h after lunch for the measurement of blood glucose, serum insulin, glucose-dependent insulinotropic polypeptide (GIP), and glucagon-like peptide 1 (GLP-1). RESULTS: The insulin responses were higher after both breakfast (31%) and lunch (57%) when whey was included in the meal than when whey was not included. After lunch, the blood glucose response was significantly reduced [-21%; 120 min area under the curve (AUC)] after whey ingestion. Postprandial GIP responses were higher after whey ingestion, whereas no differences were found in GLP-1 between the reference and test meals. CONCLUSIONS: It can be concluded that the addition of whey to meals with rapidly digested and absorbed carbohydrates stimulates insulin release and reduces postprandial blood glucose excursion after a lunch meal consisting of mashed potatoes and meatballs in type 2 diabetic subjects.

Improved stability, insulin-releasing activity and antidiabetic potential of two novel N-terminal analogues of gastric inhibitory polypeptide: N-acetyl-GIP and pGlu-GIP.

AIMS/HYPOTHESIS: This study examined the plasma stability, biological activity and antidiabetic potential of two novel N-terminally modified analogues of gastric inhibitory polypeptide (GIP). METHODS: Degradation studies were carried out on GIP, N-acetyl-GIP (Ac-GIP) and N-pyroglutamyl-GIP (pGlu-GIP) in vitro following incubation with either dipeptidylpeptidase IV or human plasma. Cyclic adenosine 3'5' monophosphate (cAMP) production was assessed in Chinese hamster lung fibroblast cells transfected with the human GIP receptor. Insulin-releasing ability was assessed in vitro in BRIN-BD11 cells and in obese diabetic ( ob/ ob) mice. RESULTS: GIP was rapidly degraded by dipeptidylpeptidase IV and plasma (t(1/2) 2.3 and 6.2 h, respectively) whereas Ac-GIP and pGlu-GIP remained intact even after 24 h. Both Ac-GIP and pGlu-GIP were extremely potent ( p<0.001) at stimulating cAMP production (EC(50) values 1.9 and 2.7 nmol/l, respectively), almost a tenfold increase compared to native GIP (18.2 nmol/l). Both Ac-GIP and pGlu-GIP (10(-13)-10(-8) mmol/l) were more potent at stimulating insulin release compared to the native GIP ( p<0.001), with 1.3-fold and 1.2-fold increases observed at 10(-8) mol/l, respectively. Administration of GIP analogues (25 nmol/kg body weight, i.p.) together with glucose (18 mmol/kg) in ( ob/ ob) mice lowered ( p<0.001) individual glucose values at 60 min together with the areas under the curve for glucose compared to native GIP. This antihyperglycaemic effect was coupled to a raised ( p<0.001) and more prolonged insulin response after administration of Ac-GIP and pGlu-GIP (AUC, 644+/-54 and 576+/-51 ng.ml(-1) x min, respectively) compared with native GIP (AUC, 257+/-29 ng.ml(-1) x min). CONCLUSION/INTERPRETATION: Ac-GIP and pGlu-GIP, show resistance to plasma dipeptidylpeptidase IV degradation, resulting in enhanced biological activity and improved antidiabetic potential in vivo, raising the possibility of their use in therapy of Type II (non-insulin-dependent) diabetes mellitus.

Degradation of endogenous and exogenous gastric inhibitory polypeptide in healthy and in type 2 diabetic subjects as revealed using a new assay for the intact peptide.

Gastric inhibitory polypeptide (GIP) is susceptible to degradation, but only recently has dipeptidyl peptidase IV been identified as the enzyme responsible. Most RIAs recognize both intact GIP-(1-42) and the noninsulinotropic N-terminally truncated metabolite, GIP-(3-42), hampering measurement of plasma concentrations. The molecular nature of GIP was examined using high pressure liquid chromatography and a newly developed RIA specific for the intact N-terminus of human GIP. In healthy subjects after a mixed meal, intact GIP (N-terminal RIA) accounted for 37.0+/-2.5% of the total immunoreactivity determined by C-terminal assay. High pressure liquid chromatographic analysis of fasting samples by C-terminal assay revealed one major peak (73.8+/-2.9%) coeluting with GIP-(3-42). One hour postprandially, two major peaks were detected, corresponding to GIP-(3-42) and GIP-(1-42) (58.1+/-2.7% and 35.7+/-4.2%, respectively). GIP-(3-42) was not detected by N-terminal assay; the major peak coeluted with intact GIP (86.4+/-5.8% and 81.3+/-0.9%, 0 and 1 h, respectively). After iv infusion, intact GIP constituted 37.1+/-4.1% and 41.3+/-3.4% of the total immunoreactivity in healthy and type 2 diabetic subjects, respectively. The plasma t1/2 was shorter (P < 0.0001) when determined by N-terminal compared with C-terminal assay (7.3+/-1.0 vs. 16.8+/-1.6 and 5.2+/-0.6 vs. 12.9+/-0.9 min, healthy and diabetic subjects, respectively), and both t1/2 were shorter in the diabetic group (P < 0.05). We conclude that dipeptidyl peptidase IV is important in GIP metabolism in humans in vivo, and that an N-terminally directed assay is required for determination of plasma concentrations of biologically active GIP.

Improved glycaemic control in obese diabetic ob/ob mice using N-terminally modified gastric inhibitory polypeptide.

Gastric inhibitory polypeptide (GIP) is an important insulin-releasing hormone of the enteroinsular axis which is rapidly inactivated by the exopeptidase dipeptidyl peptidase (DPP) IV. The present study has examined the ability of Tyr(1)-glucitol GIP to be protected from plasma degradation and to enhance insulin-releasing and antihyperglycaemic activity in 20- to 25-week-old obese diabetic ob/ob mice. Degradation of GIP by incubation at 37 degrees C with obese mouse plasma was clearly evident after 3 h (35% degraded). After 6 h, more than 61% of GIP was converted to GIP(3-42) whereas N-terminally modified Tyr(1)-glucitol GIP was resistant to degradation in plasma (>99% intact after 6 h). The formation of GIP(3-42) was almost completely abolished by inhibition of plasma DPP IV with diprotin A. Effects of GIP and Tyr(1)-glucitol GIP were examined in overnight-fasted obese mice following i.p. injection of either peptide (20 nmol/kg) together with glucose (18 mmol/kg) or in association with feeding. Most prominent effects were observed in the former group where plasma glucose values at 60 min together with the area under the curve (AUC) for glucose were significantly lower following GIP (AUC, 874+/-72 mmol/l.min; P<0.01) or Tyr(1)-glucitol GIP (770+/-134 mmol/l.min; P<0.001) as compared with administration of glucose alone (1344+/-136 mmol/l.min). This was associated with a significantly greater and more protracted insulin response following Tyr(1)-glucitol GIP than GIP (AUC, 491+/-118 vs 180+/-33 ng/ml.min; P<0.05). Administration of Tyr(1)-glucitol GIP also enhanced the glucose-lowering ability of 50 units/kg insulin (218.4+/-30.2 vs insulin alone 133.9+/-16.2 mmol/l.min; P<0.05). These data demonstrate that Tyr(1)-glucitol GIP displays resistance to plasma DPP IV degradation in a commonly used animal model of type 2 diabetes, resulting in enhanced antihyperglycaemic activity and insulin-releasing action in vivo.

Glucose-dependent insulinotropic polypeptide stimulated insulin release from a tumor-derived beta-cell line (beta TC3).

The beta TC3 tumor cell line was examined for the presence of functional glucose-dependent insulinotropic polypeptide (GIP) receptors. Increasing amounts of natural porcine GIP decreased the binding of HPLC-purified [125I]GIP to beta TC3 cells in a concentration-dependent manner. Displacement of GIP was significant at concentrations as low as 500 pM, and the radioligand was fully displaced at 100 nM. GIP(1-30) produced a displacement of [125I]GIP comparable with that produced by GIP(1-42), and glucagon yielded 20% displacement at a concentration of 1 microM but was without effect at 100 mM. Incubation of beta TC3 cells in the presence of glucose concentrations of 2-20 mM yielded a concentration-dependent stimulation of immunoreactive insulin (IRI) release. GIP and glucagon-like peptide-I(7-36) amide (tGLP-I) at concentrations of 1 nM or greater significantly stimulated IRI release in the presence of 2 mM glucose. The threshold glucose concentration for GIP-stimulated IRI release from beta TC3 cells was 0.5 mM, and maximal potentiation of IRI release by GIP occurred at 5 mM glucose. Somatostatin significantly inhibited GIP-stimulated IRI release in the presence of 5 mM glucose. It is concluded that beta TC3 cells have functional GIP receptors and may provide a useful model for the study of IRI secretion at the cellular level.

GIP and insulin release in relation to gastric emptying of a mixed meal in man.

To clarify the role of GIP (gastric inhibitory polypeptide) as an incretin, we related temporally the gastric emptying of fat, protein and glucose to plasma levels of glucose, GIP and insulin in man. Five healthy volunteers with a multiple lumen duodenal tube ingested a mixed meal with phase-specific markers for the aqueous phase, liquid fat and the solid protein phase. Duodenal passage was determined by intraduodenal infusion of a second set of phase-specific non-absorbable markers. Plasma insulin rose rapidly from a basal value of 59 pM to 300 pM at 60 min, and then declined to reach basal levels after 180 min. By contrast, plasma GIP rose more slowly than insulin, from a basal value of 9.4 pM, and remained elevated, in the range of 14-18 pM, throughout the 240 min observation period. The time course of plasma insulin concentration paralleled gastric emptying of the aqueous phase, containing most of the meal's glucose (r = 0.952, P less than 0.001). The time course of plasma GIP concentrations paralleled the gastric emptying of fat and protein (r = 0.763-0.834; P less than 0.01-0.05). Plasma insulin concentrations showed no correlation to the rate of emptying of fat and protein (r = 0.142-0.420; n.s.) and to plasma levels of GIP (r = 0.365; n.s.). The threshold for plasma glucose at which GIP would exert an incretin effect only reached at one time point, 30 min after ingestion of the meal. Our findings of simultaneously tracked gastric emptying of meal nutrients, hormone release and plasma glucose levels do not support an important physiological role for GIP as an insulinotropic hormone after ingestion of mixed meals in man.

Metabolic clearance rate and secretion rate of gastric inhibitory polypeptide in the rat.

The metabolic clearance rates (MCR) and secretion rates (SR) of immunoreactive gastric inhibitory polypeptide (IR-GIP) were determined in both fasted and fed rats by a specific radioimmunoassay (RIA). Rats were infused intravenously (iv) with porcine GIP dissolved in a blood replacement mixture at a constant rate of 0.12-0.13 ml/min for 60 min. The basal level of plasma GIP was decreased in fasted rats as compared to fed rats. The mean MCR of GIP was 1.85 ml/min in fasted rats and 1.96 ml/min in fed rats. There was no significant difference in MCR of GIP between fasted and fed rats. However, the SR was significantly higher in fed rats when compared with fasted rats. These results suggest that the low concentration of plasma GIP in fasted rats is due to a reduction of GIP secretion rate.