GIP and GLP-1 as incretin hormones: lessons from single and double incretin receptor knockout mice

Glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) are gut-derived incretins secreted in response to nutrient ingestion. Both incretins potentiate glucose-dependent insulin secretion and enhance beta-cell mass through regulation of beta-cell proliferation, neogenesis and apoptosis. In contrast, GLP-1, but not GIP, inhibits gastric emptying, glucagon secretion, and food intake. Furthermore, human subjects with Type 2 diabetes exhibit relative resistance to the actions of GIP, but not GLP-1R agonists. The physiological importance of both incretins has been investigated through generation and analysis of incretin receptor knockout mice. Elimination of incretin receptor action in GIPR-/- or GLP-1R-/- mice produces only modest impairment in glucose homeostasis. Similarly, double incretin receptor knockout (DIRKO) mice exhibit normal body weight and normal levels of plasma glucagon and hypoglycemic responses to exogenous insulin. However, glucose-stimulated insulin secretion is significantly decreased following oral but not intraperitoneal glucose challenge in DIRKO mice and the glucose lowering actions of dipeptidyl peptidase-IV (DPP-IV) inhibitors are extinguished in DIRKO mice. Hence, incretin receptor signaling exerts physiologically relevant actions critical for glucose homeostasis, and represents a pharmacologically attractive target for development of agents for the treatment of Type 2 diabetes.     

The GIP Receptor Displays Higher Basal Activity than the GLP-1 Receptor but Does Not Recruit GRK2 or Arrestin3 Effectively

Background and Objectives

Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are important regulators of insulin secretion, and their functional loss is an early characteristic of type 2 diabetes mellitus (T2DM). Pharmacological levels of GLP-1, but not GIP, can overcome this loss. GLP-1 and GIP exert their insulinotropic effects through their respective receptors expressed on pancreatic β-cells. Both the GLP-1 receptor (GLP-1R) and the GIP receptor (GIPR) are members of the secretin family of G protein-coupled receptors (GPCRs) and couple positively to adenylate cyclase. We compared the signalling properties of these two receptors to gain further insight into why GLP-1, but not GIP, remains insulinotropic in T2DM patients.

Methods

GLP-1R and GIPR were transiently expressed in HEK-293 cells, and basal and ligand-induced cAMP production were investigated using a cAMP-responsive luciferase reporter gene assay. Arrestin3 (Arr3) recruitment to the two receptors was investigated using enzyme fragment complementation, confocal microscopy and fluorescence resonance energy transfer (FRET).

Results

GIPR displayed significantly higher (P<0.05) ligand-independent activity than GLP-1R. Arr3 displayed a robust translocation to agonist-stimulated GLP-1R but not to GIPR. These observations were confirmed in FRET experiments, in which GLP-1 stimulated the recruitment of both GPCR kinase 2 (GRK2) and Arr3 to GLP-1R. These interactions were not reversed upon agonist washout. In contrast, GIP did not stimulate recruitment of either GRK2 or Arr3 to its receptor. Interestingly, arrestin remained at the plasma membrane even after prolonged (30 min) stimulation with GLP-1. Although the GLP-1R/arrestin interaction could not be reversed by agonist washout, GLP-1R and arrestin did not co-internalise, suggesting that GLP-1R is a class A receptor with regard to arrestin binding.

Conclusions

GIPR displays higher basal activity than GLP-1R but does not effectively recruit GRK2 or Arr3.     

Two incretin hormones GLP-1 and GIP: comparison of their actions in insulin secretion and β cell preservation

Gastric inhibitory polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) are the two primary incretin hormones secreted from the intestine upon ingestion of glucose or nutrients to stimulate insulin secretion from pancreatic β cells. GIP and GLP-1 exert their effects by binding to their specific receptors, the GIP receptor (GIPR) and the GLP-1 receptor (GLP-1R), which belong to the G-protein coupled receptor family. Receptor binding activates and increases the level of intracellular cAMP in pancreatic β cells, thereby stimulating insulin secretion glucose-dependently. In addition to their insulinotropic effects, GIP and GLP-1 have been shown to preserve pancreatic β cell mass by inhibiting apoptosis of β cells and enhancing their proliferation. Due to such characteristics, incretin hormones have been gaining mush attention as attractive targets for treatment of type 2 diabetes, and indeed incretin-based therapeutics have been rapidly disseminated worldwide. However, despites of plethora of rigorous studies, molecular mechanisms underlying how GIPR and GLP-1R activation leads to enhancement of glucose-dependent insulin secretion are still largely unknown. Here, we summarize the similarities and differences of these two incretin hormones in secretion and metabolism, their insulinotropic actions and their effects on pancreatic β cell preservation. We then try to discuss potential of GLP-1 and GIP in treatment of type 2 diabetes.     

Evolutionarily conserved residues at glucagon-like peptide-1 (GLP-1) receptor core confer ligand-induced receptor activation

Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) play important roles in insulin secretion through their receptors, GLP1R and GIPR. Although GLP-1 and GIP are attractive candidates for treatment of type 2 diabetes and obesity, little is known regarding the molecular interaction of these peptides with the heptahelical core domain of their receptors. These core domains are important not only for specific ligand binding but also for ligand-induced receptor activation. Here, using chimeric and point-mutated GLP1R/GIPR, we determined that evolutionarily conserved amino acid residues such as Ile(196) at transmembrane helix 2, Leu(232) and Met(233) at extracellular loop 1, and Asn(302) at extracellular loop 2 of GLP1R are responsible for interaction with ligand and receptor activation. Application of chimeric GLP-1/GIP peptides together with molecular modeling suggests that His(1) of GLP-1 interacts with Asn(302) of GLP1R and that Thr(7) of GLP-1 has close contact with a binding pocket formed by Ile(196), Leu(232), and Met(233) of GLP1R. This study may provide critical clues for the development of peptide and/or nonpeptide agonists acting at GLP1R.     

Postnatal development of numbers and mean sizes of pancreatic islets and beta-cells in healthy mice and GIPR(dn) transgenic diabetic mice

The aim of this study was to examine postnatal islet and beta-cell expansion in healthy female control mice and its disturbances in diabetic GIPR(dn) transgenic mice, which exhibit an early reduction of beta-cell mass. Pancreata of female control and GIPR(dn) transgenic mice, aged 10, 45, 90 and 180 days were examined, using state-of-the-art quantitative-stereological methods. Total islet and beta-cell volumes, as well as their absolute numbers increased significantly until 90 days in control mice, and remained stable thereafter. The mean islet volumes of controls also increased slightly but significantly between 10 and 45 days of age, and then remained stable until 180 days. The total volume of isolated beta-cells, an indicator of islet neogenesis, and the number of proliferating (BrdU-positive) islet cells were highest in 10-day-old controls and declined significantly between 10 and 45 days. In GIPR(dn) transgenic mice, the numbers of islets and beta-cells were significantly reduced from 10 days of age onwards vs. controls, and no postnatal expansion of total islet and beta-cell volumes occurred due to a reduction in islet neogenesis whereas early islet-cell proliferation and apoptosis were unchanged as compared to control mice. Insulin secretion in response to pharmacological doses of GIP was preserved in GIPR(dn) transgenic mice, and serum insulin to pancreatic insulin content in response to GLP-1 and arginine was significantly higher in GIPR(dn) transgenic mice vs. controls. We could show that the increase in islet number is mainly responsible for expansion of islet and beta-cell mass in healthy control mice. GIPR(dn) transgenic mice show a disturbed expansion of the endocrine pancreas, due to perturbed islet neogenesis.     

Tyr1 and Ile7 of glucose-dependent insulinotropic polypeptide (GIP) confer differential ligand selectivity toward GIP and glucagon-like peptide-1 receptors

Glucagon like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are incretin hormones released in response to food intake and potentiate insulin secretion from pancreatic β cells through their distinct yet related G protein-coupled receptors, GLP1R and GIPR. While GLP-1 and GIP exhibit similarity in their N-terminal sequence and overall α-helical structure, GLP-1 does not bind to GIPR and vice versa. To determine which amino acid residues of these peptide ligands are responsible for specific interaction with their respective receptors, we generated mutant GIP in which several GLP-1-specific amino acid residues were substituted for the original amino acids. The potency of the mutant ligands was examined using HEK293 cells transfected with GLP1R or GIPR expression plasmids together with a cAMP-responsive element-driven luciferase (CRE-luc) reporter plasmid. A mutated GIP peptide in which Tyr¹, Ile⁷, Asp¹⁵, and His¹⁸ were replaced by His, Thr, Glu, and Ala, respectively, was able to activate both GLP1R and GIPR with moderate potency. Replacing the original Tyr¹ and/or Ile⁷ in the N-terminal moiety of this mutant peptide allowed full activation of GIPR but not of GLP1R. However, reintroducing Asp¹⁵ and/or His¹⁸ in the central α-helical region did not significantly alter the ligand potency. These results suggest that Tyr/His¹ and Ile/Thr⁷ of GIP/GLP-1 peptides confer differential ligand selectivity toward GIPR and GLP1R.     

GIP biology and fat metabolism

The gastrointestinal hormone, gastric inhibitory polypeptide (GIP), is synthesized and released from the duodenum and proximal jejunum postprandially. Its release depends upon several factors including meal content and pre-existing health status (ie. obesity, diabetes, age, etc.). It was initially discovered and named for its gastric acid inhibitory properties. However, its more physiologically relevant role appears to be as an insulinotropic agent with a stimulatory effect on insulin release and synthesis. Accordingly, it was later renamed glucose-dependent insulinotropic polypeptide because its action on insulin release depends upon an increase in circulating levels of glucose. GIP is considered to be one of the principle incretin factors of the enteroinsular axis. The GIP receptor is a G-protein-coupled receptor belonging to the family of secretin/VIP receptors. GIP receptor mRNA is widely distributed in peripheral organs, including the pancreas, gut, adipose tissue, heart, adrenal cortex, and brain, suggesting it may have other functions in addition to the ones mentioned above. An overactive enteroinsular axis has been suggested to play a role in the pathogenesis of diabetes and obesity. In addition to stimulating insulin release, GIP has been shown to amplify the effect of insulin on target tissues. In adipose tissue, GIP has been reported to (1) stimulate fatty acid synthesis, (2) enhance insulin-stimulated incorporation of fatty acids into triglycerides, (3) increase insulin receptor affinity, and (4) increase sensitivity of insulin-stimulated glucose transport. In addition, although controversial, lipolytic properties of GIP have been proposed. The mechanism of action of GIP-induced effects on adipocytes is unknown, and it is unclear whether these effects of GIP on adipocytes are direct or indirect. However, there is now evidence that GIP receptors are expressed on adipocytes and that these receptors respond to GIP stimulation. Given the location of its release and the timing of its release, GIP is an ideal anabolic agent and expanding our understanding of its physiology will be needed to determine its exact role in the etiology of diabetes mellitus and obesity.     

Glucose-dependent insulinotropic polypeptide (GIP) stimulation of pancreatic beta-cell survival is dependent upon phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB) signaling, inactivation of the forkhead transcription factor Foxo1, and down-regulation of bax expression

The hormone glucose-dependent insulinotropic polypeptide (GIP) potently stimulates insulin secretion and promotes beta-cell proliferation and cell survival. In the present study we identified Forkhead (Foxo1)-mediated suppression of the bax gene as a critical component of the effects of GIP on cell survival. Treatment of INS-1(832/13) beta-cells with GIP resulted in concentration-dependent activation of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB)/Foxo1 signaling module. In parallel studies, GIP decreased bax promoter activity. Serial deletion analysis of the bax promoter demonstrated that the region -682 to -320, containing FHRE-II (5AAAACAAACA), was responsible for GIP-mediated effects. Foxo1 bound to FHRE-II in gel mobility shift assays, and Foxo1-FHRE-II interactions conferred GIP responsiveness to the bax promoter. INS-1 cells incubated under proapoptotic and glucolipotoxic conditions demonstrated increased nuclear localization of Foxo1 and bax promoter activity and decreased cytoplasmic phospho-PKB/Foxo1. GIP partially restored expression PKB/Foxo1 and bax promoter activity. Similar protective effects were found with dispersed islet cells from C57BL/6 mice, but not with those from GIP receptor knock-out (GIPR(-/-)) mice. GIP treatment reduced glucolipotoxicity-induced cell death in C57 BL/6 and Bax(-/-) islets, but not GIPR(-/-) mouse islets. Chronic treatment of Vancouver diabetic fatty Zucker rats with GIP resulted in down-regulation of Bax and up-regulation of Bcl-2 in pancreatic beta-cells. The results show that PI3K/PKB/Foxo1 signaling mediates GIP suppression of bax gene expression and that this module is a key pathway by which GIP regulates beta-cell apoptosis in vivo.     

Incretin and islet hormonal responses to fat and protein ingestion in healthy men

Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) regulate islet function after carbohydrate ingestion. Whether incretin hormones are of importance for islet function after ingestion of noncarbohydrate macronutrients is not known. This study therefore examined integrated incretin and islet hormone responses to ingestion of pure fat (oleic acid; 0.88 g/kg) or protein (milk and egg protein; 2 g/kg) over 5 h in healthy men, aged 20-25 yr (n=12); plain water ingestion served as control. Both intact (active) and total GLP-1 and GIP levels were determined as was plasma activity of dipeptidyl peptidase-4 (DPP-4). Following water ingestion, glucose, insulin, glucagon, GLP-1, and GIP levels and DPP-4 activity were stable during the 5-h study period. Both fat and protein ingestion increased insulin, glucagon, GIP, and GLP-1 levels without affecting glucose levels or DPP-4 activity. The GLP-1 responses were similar after protein and fat, whereas the early (30 min) GIP response was higher after protein than after fat ingestion (P<0.001). This was associated with sevenfold higher insulin and glucagon responses compared with fat ingestion (both P<0.001). After protein, the early GIP, but not GLP-1, responses correlated to insulin (r(2)=0.86; P=0.0001) but not glucagon responses. In contrast, after fat ingestion, GLP-1 and GIP did not correlate to islet hormones. We conclude that, whereas protein and fat release both incretin and islet hormones, the early GIP secretion after protein ingestion may be of primary importance to islet hormone secretion.     

The biology of incretin hormones

Gut peptides, exemplified by glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are secreted in a nutrient-dependent manner and stimulate glucose-dependent insulin secretion. Both GIP and GLP-1 also promote β cell proliferation and inhibit apoptosis, leading to expansion of β cell mass. GLP-1, but not GIP, controls glycemia via additional actions on glucose sensors, inhibition of gastric emptying, food intake and glucagon secretion. Furthermore, GLP-1, unlike GIP, potently stimulates insulin secretion and reduces blood glucose in human subjects with type 2 diabetes. This article summarizes current concepts of incretin action and highlights the potential therapeutic utility of GLP-1 receptor agonists and dipeptidyl peptidase-4 (DPP-4) inhibitors for the treatment of type 2 diabetes.     

Priming effect of glucagon-like peptide-1 (7-36) amide, glucose-dependent insulinotropic polypeptide and cholecystokinin-8 at the isolated perfused rat pancreas.

The priming effect of glucagon-like peptide-1 (7-36) amide (GLP-1 (7-36) amide), glucose-dependent insulin-releasing polypeptide (GIP) and cholecystokinin-8 (CCK-8) on glucose-induced insulin secretion from rat pancreas was investigated. The isolated pancreas was perfused in vitro with Krebs-Ringer bicarbonate buffer containing 2.8 mmol/l glucose. After 10 min this medium was supplemented with GLP-1 (7-36) amide, GIP or CCK-8 (10, 100, 1000 pmol/l) for 10 min. After an additional 10 min period with 2.8 mmol/l glucose alone, insulin secretion was stimulated with buffer containing 10 mmol/l glucose for 44 min. In control experiments the typical biphasic insulin response to 10 mmol/l glucose occurred. Pretreatment of the pancreas with GIP augmented insulin secretion: 10 pmol/l GIP enhanced only the first phase of the secretory response to 10 mmol/l glucose; 100 and 1000 pmol/l GIP stimulated both phases of hormone secretion. After exposure to CCK-8, enhanced insulin release during the first (at 10 and 1000 pmol/l CCK-8) and the second phase (at 1000 pmol/l) was observed. Priming with 100 pmol/l GLP-1 (7-36) amide significantly amplified the first and 1000 pmol/l GLP-1 (7-36) amide both secretion periods, 10 pmol/l GLP-1 (7-36) amide had no significant effect. All three peptide hormones influenced the first, quickly arising secretory response more than the second phase. Priming with forskolin (30 mM) enhanced the secretory response to 10 mM glucose plus 0.5 nM GLP-1 (7-36) amide 4-fold. With a glucose-responsive B-cell line (HIT cells), we investigated the hypothesis that the priming effect of GLP-1 (7-36) amide is mediated by the adenylate cyclase system. Priming with either IBMX (0.1 mM) or forskolin (2.5 microM) enhanced the insulin release after a consecutive glucose stimulation (5 mM). This effect was pronounced when GLP-1 (7-36) amide (100 pM) was added during glucose stimulation. Priming capacities of intestinal peptide hormones may be involved in the regulation of postprandial insulin release. The incretin action of these hormones can probably, at least in part, be explained by these effects. The priming effect of GLP-1 (7-36) amide is most likely mediated by the adenylate cyclase system.     

Acute activation of central GLP-1 receptors enhances hepatic insulin action and insulin secretion in high-fat-fed, insulin resistant mice

Glucagon-like peptide-1 (GLP-1) receptor knockout (Glp1r(-/-)) mice exhibit impaired hepatic insulin action. High fat (HF)-fed Glp1r(-/-) mice exhibit improved, rather than the expected impaired, hepatic insulin action. This is due to decreased lipogenic gene expression and triglyceride accumulation. The present studies overcome these secondary adaptations by acutely modulating GLP-1R action in HF-fed wild-type mice. The central GLP-1R was targeted given its role as a regulator of hepatic insulin action. We hypothesized that acute inhibition of the central GLP-1R impairs hepatic insulin action beyond the effects of HF feeding. We further hypothesized that activation of the central GLP-1R improves hepatic insulin action in HF-fed mice. Insulin action was assessed in conscious, unrestrained mice using the hyperinsulinemic euglycemic clamp. Mice received intracerebroventricular (icv) infusions of artificial cerebrospinal fluid, GLP-1, or the GLP-1R antagonist exendin-9 (Ex-9) during the clamp. Intracerebroventricular Ex-9 impaired the suppression of hepatic glucose production by insulin, whereas icv GLP-1 improved it. Neither treatment affected tissue glucose uptake. Intracerebroventricular GLP-1 enhanced activation of hepatic Akt and suppressed hypothalamic AMP-activated protein kinase. Central GLP-1R activation resulted in lower hepatic triglyceride levels but did not affect muscle, white adipose tissue, or plasma triglyceride levels during hyperinsulinemia. In response to oral but not intravenous glucose challenges, activation of the central GLP-1R improved glucose tolerance. This was associated with higher insulin levels. Inhibition of the central GLP-1R had no effect on oral or intravenous glucose tolerance. These results show that inhibition of the central GLP-1R deteriorates hepatic insulin action in HF-fed mice but does not affect whole body glucose homeostasis. Contrasting this, activation of the central GLP-1R improves glucose homeostasis in HF-fed mice by increasing insulin levels and enhancing hepatic insulin action.     

Effect of cholecystokinin, secretin, and gastric inhibitory polypeptide on insulin release from the isolated perfused rat pancreas

The actions of gastric inhibitory polypeptide (GIP) on insulin release from the isolated perfused rat pancreas were compared with those of pure secretin and cholecystokinin (CCK). At dose levels physiologically achievable for GIP (1 ng/mL perfusate), infusions of CCK stimulated significant insulin release both on a weight (1 ng/mL) and a molar (770 pg/mL) basis. Although 50% as potent as GIP on a weight basis and 43% as potent on a molar basis, the insulin response to CCK was multiphasic and sustained for the duration of the infusion. The action of CCK, like that of GIP, was glucose dependent yielding no significant insulin release at a low perfusate glucose concentration (80 mg/dL). Irrespective of perfusate glucose concentration or dose (1 or 5 ng/mL), secretin failed to stimulate significant release of insulin from the perfused pancreas. It was concluded that secretin is ineffective as an incretin and that a physiological role for CCK in an enteroinsular axis awaits accurate measurement of circulating levels of immunoreactive CCK.     

A Novel Glucagon-like Peptide-1 (GLP-1)/Glucagon Hybrid Peptide with Triple-acting Agonist Activity at Glucose-dependent Insulinotropic Polypeptide,GLP-1, and Glucagon Receptors and Therapeutic Potential in High Fat-fed Mice

Glucagon-like peptide-1 (GLP-1), glucose-dependent insulinotropic polypeptide (GIP), and glucagon bind to related members of the same receptor superfamily and exert important effects on glucose homeostasis, insulin secretion, and energy regulation. The present study assessed the biological actions and therapeutic utility of novel GIP/glucagon/GLP-1 hybrid peptides. Nine novel peptides were synthesized and exhibited complete DPP-IV resistance and enhanced in vitro insulin secretion. The most promising peptide, [dA²]GLP-1/GcG, stimulated cAMP production in GIP, GLP-1, and glucagon receptor-transfected cells. Acute administration of [dA²]GLP-1/GcG in combination with glucose significantly lowered plasma glucose and increased plasma insulin in normal and obese diabetic (ob/ob) mice. Furthermore, [dA²]GLP-1/GcG elicited a protracted glucose-lowering and insulinotropic effect in high fat-fed mice. Twice daily administration of [dA²]GLP-1/GcG for 21 days decreased body weight and nonfasting plasma glucose and increased circulating plasma insulin concentrations in high fat-fed mice. Furthermore, [dA²]GLP-1/GcG significantly improved glucose tolerance and insulin sensitivity by day 21. Interestingly, locomotor activity was increased in [dA²]GLP-1/GcG mice, without appreciable changes in aspects of metabolic rate. Studies in knock-out mice confirmed the biological action of [dA²]GLP-1/GcG via multiple targets including GIP, GLP-1, and glucagon receptors. The data suggest significant promise for novel triple-acting hybrid peptides as therapeutic options for obesity and diabetes.     

Incretin hormone receptors are required for normal beta cell development and function in female mice

The incretin hormones, glucose dependent insulinotropic polypeptide (GIP) and glucagon-like peptide 1 (GLP-1), potentiate insulin secretion and are responsible for the majority of insulin secretion that occurs after a meal. They may also, however, have a fundamental role in pancreatic beta cell development and function, independently of their role in potentiating insulin secretion after a meal. This has led to observations that a loss of GIP or GLP-1 action affects normal beta cell function, however each one of the incretin hormones may compensate when the action of the other is lost and therefore the overall impact of the incretin hormones on beta cell function is not known. We therefore utilized a mouse line deficient in both the GLP-1 and GIP receptor genes, the double incretin receptor knockout (DIRKO), to determine the consequences of a lifelong, complete lack of incretin hormone action on beta cell function, in vivo, in intact animals. We found that DIRKO mice displayed impaired glucose tolerance and insulin secretion in response to both oral glucose and mixed meal tolerance tests compared to wild-type mice. Assessment of beta cell function using the hyperglycemic clamp technique revealed an 80% decrease in first phase insulin response in DIRKO mice, but a normal second phase insulin secretion. A similar decline was seen when wild-type mice were given acute intravenous injection of glucose together with the GLP-1 receptor antagonist Ex9-39. Ex vivo assessments of the pancreas revealed significantly fewer islets in the pancreata of DIRKO mice despite no differences in total pancreatic mass. Insulin secretion from isolated islets of DIRKO mice was impaired to a similar extent to that seen during the hyperglycemic clamp. Insulin secretion in wild-type islets was impaired by acute treatment with Ex9-39 to a similar extent as the in vivo intravenous glucose tolerance tests. In conclusion, a loss of the action of both incretin hormones results in direct impairment of beta cell function both in vivo and in vitro in a process that appears to be independent of the intestinally secreted incretin hormones. We therefore conclude that the incretin hormones together significantly impact both beta-cell function and beta-cell development.     

Simultaneous determination of incretin hormones and their truncated forms from human plasma by immunoprecipitation and liquid chromatography-mass spectrometry

The incretins, glucose-dependent insulinotropic peptide (GIP(1-42)) and glucagon-like peptide 1 (GLP-1(7-36)), are involved in regulation of gastric emptying, glucose homeostasis, body fat regulation and the glucose-induced insulin secretion from the endocrine pancreas. After release in the circulation both peptides are rapidly degraded by the exopeptidase dipeptidyl peptidase IV (DP IV) to the inactive polypeptides GIP(3-42) and GLP-1(9-36). In vivo stabilization of the active incretins by orally available DP IV-inhibitors is now widely accepted as a new therapeutic approach in antidiabetic treatment. In order to demonstrate the pharmacodynamic effect of DP IV-inhibitors, it is necessary to measure the plasma levels of active and inactive forms of GIP and GLP-1. We previously described an immunoprecipitation method as sample preparation and concentration in combination with a LC-MS analysis for determination of active and inactive GIP. We could improve the efficiency and suitability of this method by reduction of the necessary sample volume to 1.0 ml and simultaneous measurement of GIP(1-42), GIP(3-42) and GLP-1(7-36), GLP-1(9-36), without loss of sensitivity. An LOQ of approximately 5 and 11 pmol/l was maintained for GIP and GLP-1, respectively.     

Actions of incretin metabolites on locomotor activity,cognitive function and in vivo hippocampal synaptic plasticity in high fat fed mice

The incretin hormones glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) improve markers of cognitive function in obesity–diabetes, however, both are rapidly degraded to their major metabolites, GLP-1(9-36)amide and GIP(3-42), respectively. Therefore, the present study investigated effects of GLP-1(9-36)amide and GIP(3-42) on locomotor activity, cognitive function and hippocampal synaptic plasticity in mice with diet-induced obesity and insulin resistance. High-fat fed Swiss TO mice treated with GLP-1(9-36)amide, GIP(3-42) or exendin(9-39)amide (twice-daily for 60 days) did not exhibit any changes in bodyweight, non-fasting plasma glucose and plasma insulin concentrations or glucose tolerance compared with high-fat saline controls. Similarly, locomotor and feeding activity, O₂ consumption, CO₂ production, respiratory exchange ratio and energy expenditure were not altered by chronic treatment with incretin metabolites. Administration of the truncated metabolites did not alter general behavior in an open field test or learning and memory ability as recorded during an object recognition test. High-fat mice exhibited a significant impairment in hippocampal long-term potentiation (LTP) which was not affected by treatment with incretin metabolites. These data indicate that incretin metabolites do not influence locomotor activity, cognitive function and hippocampal synaptic plasticity when administered at pharmacological doses to mice fed a high-fat diet.     

A novel GIP receptor splice variant influences GIP sensitivity of pancreatic beta-cells in obese mice

Gastric inhibitory polypeptide (GIP) is an incretin that potentiates insulin secretion from pancreatic beta-cells by binding to GIP receptor (GIPR) and subsequently increasing the level of intracellular adenosine 3',5'-cyclic monophosphate (cAMP). We have identified a novel GIPR splice variant in mouse beta-cells that retains intron 8, resulting in a COOH-terminal truncated form (truncated GIPR). This isoform was coexpressed with full-length GIPR (wild-type GIPR) in normal GIPR-expressing tissues. In an experiment using cells transfected with both GIPRs, truncated GIPR did not lead to cAMP production induced by GIP but inhibited GIP-induced cAMP production through wild-type GIPR (n = 3-4, P < 0.05). Wild-type GIPR was normally located on the cell surface, but its expression was decreased in the presence of truncated GIPR, suggesting a dominant negative effect of truncated GIPR against wild-type GIPR. The functional relevance of truncated GIPR in vivo was investigated. In high-fat diet-fed obese mice (HFD mice), blood glucose levels were maintained by compensatory increased insulin secretion (n = 8, P < 0.05), and cAMP production (n = 6, P < 0.01) and insulin secretion (n = 10, P < 0.05) induced by GIP were significantly increased in isolated islets, suggesting hypersensitivity of the GIPR. Total GIPR mRNA expression was not increased in the islets of HFD mice, but the expression ratio of truncated GIPR to total GIPR was reduced by 32% compared with that of control mice (n = 6, P < 0.05). These results indicate that a relative reduction of truncated GIPR expression may be involved in hypersensitivity of GIPR and hyperinsulinemia in diet-induced obese mice.     

Overexpression of a dominant negative GIP receptor in transgenic mice results in disturbed postnatal pancreatic islet and beta-cell development

The expression of a dominant negative glucose-dependent insulinotropic polypeptide receptor (GIPRdn) under the control of the rat pro-insulin gene promoter induces severe diabetes mellitus in transgenic mice. This study aims to gain further insight into the effect of the expression of a dominant negative GIPR on glucose homeostasis and postnatal development of the endocrine pancreas. The diabetic phenotype of GIPRdn transgenic animals was first observed between 14 and 21 days of age (urine glucose>1000 mg/dl). After onset of diabetes, serum glucose was significantly higher and insulin values were significantly lower in GIPRdn transgenic mice vs. non-transgenic littermate controls. Morphometric studies of pancreatic islets and their endocrine cell types were carried out at 10, 30 and 90 days of age. The total islet and total beta-cell volume of transgenic mice was severely reduced as compared to control mice, irrespective of the age at sampling (p<0.05). The total volume of isolated insulin positive cells that were not contained within established islets was significantly reduced in transgenic mice, indicating disturbed islet neogenesis. These findings demonstrate in vivo evidence that intact signaling of G-protein coupled receptors is involved in postnatal islet and beta-cell development and neogenesis of the pancreatic islets.