The role of GIP and pancreatic GLP-1 in the glucoregulatory effect of DPP-4 inhibition in mice
Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are two peptides that function to promote insulin secretion. Dipeptidyl peptidase-4 (DPP-4) inhibitors increase the bioavailability of both GLP-1 and GIP but the dogma continues to be that it is the increase in GLP-1 that contributes to the improved glucose homeostasis. We have previously demonstrated that pancreatic rather than intestinal GLP-1 is necessary for improvements in glucose homeostasis in mice. Therefore, we hypothesise that a combination of pancreatic GLP-1 and GIP is necessary for the full effect of DPP-4 inhibitors on glucose homeostasis.
We have genetically engineered mouse lines in which the preproglucagon gene (Gcg) is absent in the entire body (GcgRAΔNull) or is expressed exclusively in the intestine (GcgRAΔVilCre) or pancreas and duodenum (GcgRAΔPDX1Cre). These mice were used to examine oral glucose tolerance and GLP-1 and GIP responses to a DPP-4 inhibitor alone, or in combination with incretin receptor antagonists.
Administration of the DPP-4 inhibitor, linagliptin, improved glucose tolerance in GcgRAΔNull mice and control littermates and in GcgRAΔVilCre and GcgRAΔPDX1Cre mice. The potent GLP-1 receptor antagonist, exendin-[9–39] (Ex9), blunted improvements in glucose tolerance in linagliptin-treated control mice and in GcgRAΔPDX1Cre mice. Ex9 had no effect on glucose tolerance in linagliptin-treated GcgRAΔNull or in GcgRAΔVilCre mice. In addition to GLP-1, linagliptin also increased postprandial plasma levels of GIP to a similar degree in all genotypes. When linagliptin was co-administered with a GIP-antagonising antibody, the impact of linagliptin was partially blunted in wild-type mice and was fully blocked in GcgRAΔNull mice.
Taken together, these data suggest that increases in pancreatic GLP-1 and GIP are necessary for the full effect of DPP-4 inhibitors on glucose tolerance.
KeywordsDPP-4 inhibitor GIP GLP-1 Glucose homeostasis Incretin
Gcg null mouse model
Mouse model with Gcg reactivated in the pancreas
Mouse model with Gcg reactivated in the intestine
Glucose-dependent insulinotropic polypeptide
GIP receptor-antagonising antibody
Pancreatic duodenal homeobox-1
Wild-type littermate of GcgRAΔPDX1Cre
Wild-type littermate of GcgRAΔVilCre
Glucagon-like peptide-1 (GLP-1), encoded as a fragment of proglucagon in the preproglucagon gene (Gcg), is expressed in the brain, pancreatic alpha cells and enteroendocrine cells within the intestine. GLP-1 is thought to act in the central nervous system to regulate feeding behaviour and on pancreatic beta cell GLP-1 receptors (GLP-1Rs) to stimulate insulin secretion. Two potent therapeutic strategies based on the GLP-1 system have been developed for the treatment of type 2 diabetes . The first includes injectable long-acting GLP-1R agonists and the second increases the bioavailability of endogenous GLP-1 by pharmacologically inhibiting dipeptidyl peptidase-4 (DPP-4), the enzyme that removes the N-terminal amino acid and consequently inactivates GLP-1 .
While DPP-4 is important for regulating circulating active GLP-1 levels by limiting the half-life to 1–2 min , it also acts on other peptides involved in metabolic regulation including glucose-dependent insulinotropic polypeptide (GIP) . A previous study in mice suggests that increasing the bioavailability of both GIP and GLP-1 contributes to the ability of DPP-4 inhibitors to improve glucose tolerance . However, the lack of efficacy of GIP agonists in stimulating insulin secretion and promoting glucose disposal in individuals with type 2 diabetes [6, 7] raises doubts about whether both GLP-1 and GIP mediate the therapeutic effects of DPP-4 inhibitors in humans . Thus, the standard explanation as to how DPP-4 inhibitors exert their therapeutic effects is their promotion of GLP-1 signalling. However, recent clinical data suggest that elevation in plasma GLP-1 does not account for all the glucoregulatory effects of DPP-4 inhibitors .
Using Cre/loxP technology, we have generated a novel genetic mouse model that allows for the reactivation of endogenous Gcg with tissue specificity. Using this model we have found that proglucagon products produced in pancreatic alpha cells, rather than intestinal L cells, are the principle mediator of glucose tolerance . Thus, the aim of this study was to determine whether pancreas-derived or intestine-derived GLP-1 is the predominant source of the plasma GLP-1 that increases with DPP-4 inhibitors and that, in combination with an increase in GIP, drives the improvements in glucose regulation.
We developed a mouse model with a loxP flanked transcriptional blocking cassette inserted between exons 2 and 3 of the Gcg gene, as described previously . These mice were extensively phenotyped in a previous publication . The resulting mouse is null for Gcg expression (GcgRAΔNull) and therefore does not produce any proglucagon peptides (including GLP-1 and glucagon) . In some studies GcgRAΔNull mice and wild-type littermates were used as controls. In other studies we crossed the GcgRAΔNull mice to lines expressing Cre recombinase with either the villin-1 promoter (VilCre; Stock # 004586, Jackson Laboratories, Bar Harbor, ME, USA) or pancreatic duodenal homeobox-1 (PDX1Cre; Stock # 014647, Jackson Laboratories) promoter generated mouse lines with endogenous Gcg reactivated specifically in intestinal epithelial L cells (GcgRAΔVilCre) and pancreatic alpha cells and within the duodenum (GcgRAΔPDX1Cre), respectively . Both the VilCre and PDX1Cre promotors allow for reactivation of the duodenum. However, since the intestinal reactivation, which also includes the duodenum, and the pancreatic/duodenal reactivation have very different responses, then the duodenum is not a major contributor to this process, and the GcgRAΔPDX1Cre mouse is considered to be a pancreatic reactivation model for the purposes of this study. All reactivated mice were compared with littermate mice expressing Cre alone (VilCre or PDX1Cre, respectively). All mice were male on a mixed background, were at least 8 weeks old and were individually housed and maintained on a 12 h light–dark cycle (lights off at 17:00 hours) at 25°C and 50–60% humidity with ad libitum access to water and standard chow diet. All procedures for animal use were approved by the University of Michigan and University of Cincinnati Institutional Animal Care and Use Committee.
Effect of acute DPP-4 inhibition on glucose tolerance
Mice were fasted for 4–5 h prior to OGTTs. Baseline glucose levels were taken before two doses of linagliptin (3 or 30 mg/kg) were administered by gavage 30 min prior to an oral glucose load (100 μl of 20%, wt/vol. in water, dextrose). Because both doses were effective at lowering glucose, we chose an intermediate dose (10 mg/kg) of linagliptin for the remaining studies. In all experiments 0.5% natrosol (wt/vol. in water; 9004-62-0; Millipore Sigma, St Louis, MO, USA) was used as the vehicle for linagliptin. In separate studies, baseline glucose was measured and then mice were given an i.p. injection of 50 μg/100 μl of exendin-[9–39] (Ex9) (Bachem Bioscience, Torrance, CA, USA) or saline (NaCl 154 mmol/l) 45 min before an oral glucose load (200 μl of 20% wt/vol. in water, glucose) and 15 min before oral linagliptin (10 mg/kg). Blood was sampled from the tail vein at baseline and at −15, 15, 30, 45, 60 and 120 min after glucose. To study the role of GIP in the response to linagliptin, mice were administered a GIP receptor-antagonising antibody (GIPrAb; obtained from Boehringer Ingelheim, Ingelheim am Rhein, Germany). The structure of this compound and the protocol of administration was as reported previously . Briefly, mice were administered the GIPrAb (30 μg/kg), fasted for 24 h, then linagliptin (10 mg/kg) or vehicle was administered prior to an oral glucose (2 g/kg) tolerance test. Blood was sampled from the tail vein at baseline and at 0, 10, 20, 40, 60 and 120 min post glucose gavage.
When appropriate, mice were utilised in a cross-over design whereby each animal was exposed to vehicle or treatment conditions in distinct experiments with each experiment separated by at least 1 week. All mice were randomised to groups, and experimenters were blind to groups during blood glucose measurements. However, for all experiments each animal was studied under a specific drug condition once (i.e. data are from distinct samples). Animals were excluded from an analysis if the handler noted a bad injection of drug and/or glucose or if they lost weight and became ill between experiments.
Effect of chronic DPP-4 inhibition on glucose tolerance
In a separate cohort, mice were fed a high-fat diet (19 kJ [4.54 kcal]/g, 41% fat; Research Diets, New Brunswick, NJ, USA) for 16 weeks. The DPP-4 inhibitor vildagliptin was given by i.p. injection at a dose (150 μg/200 μl; Millipore Sigma, St Louis, MO, USA) equipotent (data not shown) to 10 mg/kg linagliptin, daily for 11 days. A control group was administered with saline. Body weight and food intake was measured daily and on day 11 body composition was assessed and an OGTT was performed.
Mice were administered either linagliptin (10 mg/kg) or vehicle (0.5% natrosol) via oral gavage 30 min before oral glucose (200 μl, 50%, wt/vol. in water, glucose) and 45 min prior to blood collection. Blood was collected in heparinised syringes and placed in a tube with a mixture of DPP-4 inhibitor (Millipore, Burlington, MA, USA), heparin, EDTA and aprotonin. Plasma was assayed for active GLP-1 to validate genotyping using sandwich ELISA kits (K150JWC-1; Mesoscale Discovery, Rockville, MD, USA). Plasma was assayed for active mouse GIP using an ELISA (27702; IBL America, Minneapolis, MN, USA). Plasma insulin was analysed using a standard ELISA kit (90080; Crystal Chem, Elk Grove Village, IL, USA). DPP-4 activity was measured using a specific peptide substrate with a terminal coumarin derivative (H-Ala-Pro-7-amido-4-trifluoromethylcoumarin; Bachem, Bubendorf, Switzerland), allowing quantification in a fluorescence microplate reader (Wallac Victor 1420 Multilabel Counter; Perkin Elmer, Waltham, MA, USA) upon cleavage by DPP-4 .
Mouse whole pancreas and epithelial scrapes from duodenum and ileum were immediately homogenised in Trizol (Thermo Fisher Scientific, Waltham, MA, USA) using a Tissuelyser II (Qiagen, Hilden, Germany). Total RNA was extracted using the TaqMan Reverse Transcription kit (Thermo Fisher Scientific). The quality and concentration of the isolated total RNA were determined using a Nanodrop spectrophotometer (Thermo Fisher Scientific). cDNA was isolated (iScript cDNA synthesis kit; BioRad, Hercules, CA, USA) and real-time quantitative PCR (qPCR) was performed using a TaqMan 7900 Sequence Detection System with TaqMan Universal PCR Master Mix and TaqMan Gene Expression Assays (Applied Biosystems, Foster City, CA, USA). mRNA expression was evaluated using the TaqMan gene probe mouse Gcg (Mm01269054_m1) and normalised to the ribosomal RPL32 gene (Rn00820748_g1).
Data are presented as mean ± SD (bar graphs) or mean ± SEM (line graphs), and statistical significance was set at p < 0.05 for all analyses. The data were analysed using mixed-model ANOVAs to evaluate significant main effects of drug, genotype or time (where appropriate) and significant interactions among these independent variables. Where appropriate a Tukey’s post hoc analysis was performed to determine where the significant differences lie. Because of the complex statistical design (drug, genotype, time) and potent effect of both the drug and genotype, sometimes there was no interaction with time (main effect of drug and/or genotype). These main effects are indicated in the text and figure legends. Statistical interactions are represented by symbols within figures.
Gcg peptides are not necessary for glucose-lowering effect of DPP-4 inhibitors
As we have found previously , GcgRA∆Null and GcgRA∆VilCre mice displayed improved glucose tolerance. Our previous work concluded that this was a consequence of glucagon deficiency rather than improved insulin sensitivity or secretion, as determined by a hyperinsulinaemic–euglycaemic clamp and an IVGTT . Here, we found that linagliptin significantly improved glucose tolerance at doses of 3 mg/kg and 30 mg/kg in VilCre control, GcgRA∆VilCre and GcgRA∆Null mice at 15, 30, 45 and 60 min after an oral glucose gavage (Fig. 1d–h).
Tissue-specific source of GLP-1 after DPP-4 inhibition
We then assessed the insulin response to linagliptin (10 mg/kg) after oral glucose administration in all mice. No differences in baseline insulin levels were detected across genotypes (ESM Fig. 1a). Linagliptin did not have any additional impact on the insulin response 5 or 15 min after a glucose load (ESM Fig. 1b,c).
Unlike mice with only intestinal Gcg production, Ex9 impaired oral glucose tolerance in GcgRAΔPDX1Cre and control PDX1Cre mice (Fig. 3f–h) and was able to lessen the glucose-lowering effect of linagliptin in both PDX1Cre control and GcgRAΔPDX1Cre mice. These data suggest that Gcg expression and GLP-1 synthesis within the pancreas is necessary for the glucoregulatory role of DPP-4 inhibitors.
The role of GIP in response to DPP-4 inhibition
Previous studies have suggest that GIP signalling is muted during obesity and/or type 2 diabetes [7, 14]. Based on this observation, DPP-4 inhibitors may be less effective in high-fat-diet-fed GcgRA∆Null and GcgRA∆VilCre mice that depend on GIP to mediate DPP-4 inhibitory effects. We administered vildagliptin, another DPP-4 inhibitor, to mice after 16 weeks of high-fat diet. Vildagliptin did not affect fat mass or food intake over 11 days of treatment (data not shown). Despite the prolonged exposure to high-fat diet, VilCre control, GcgRA∆Null and GcgRA∆VilCre mice all showed improved oral glucose tolerance in response to vildagliptin (Fig. 4c and ESM Fig. 2b).
DPP-4 inhibitors are effective and widely used therapeutics for type 2 diabetes. The conventionally accepted mechanism of action underlying the efficacy of DPP-4 inhibitors is that they improve glucose homeostasis through increased gut-derived plasma GLP-1 concentrations. However, preclinical work and recent clinical studies suggest that this view may be incomplete and ignores potentially important contributions from GIP. We have recently generated a transgenic mouse line that allows for tissue-selective expression of the endogenous Gcg gene . Using this model, we determined that pancreatic but not intestinal GLP-1 was the critical peptide pool necessary for regulation of normal glucose tolerance. In addition, studying the impact of DPP-4 inhibitors in combination with GLP-1 and GIP receptor antagonists in our Gcg-null animals allowed us to determine the relative roles of these two incretin peptides. Consistent with previous work [15, 16], we found that in the absence of GLP-1 production, GIP is sufficient to maintain improved glucose tolerance after DPP-4 inhibitor administration. Moreover, our findings indicate that the endocrine pancreas is also an important source of GLP-1 targeted by DPP-4 inhibitors to confer better glucose tolerance.
The proteolytic actions of DPP-4 target approximately 40 currently known substrates, with additional substrates continually being discovered . Despite this growing list, many of these compounds are primarily in vitro substrates with little evidence supporting the in vivo regulation of their activity by DPP-4 . Currently, the best evidence supports GLP-1 and GIP as important targets of DPP-4’s action in animals and humans that can be tied to physiological outcomes. Indeed, an early formulation of a DPP-4 inhibitor improved oral glucose tolerance in GLP-1R knockout mice and in GIP receptor knockout mice . However, double knockout of both GLP-1 and GIP receptors did not provide any significant improvement in oral glucose tolerance following DPP-4 inhibitor administration [15, 16]. Two recent studies that included individuals with and without type 2 diabetes found that GLP-1 contributed ~50% of the glucose-lowering and insulin-stimulatory effects of DPP-4 inhibitors [2, 18]. Using both genetic and pharmacological inhibition, our work is consistent with these data in that linagliptin retained the ability to improve glucose tolerance in animals devoid of all Gcg peptides.
Recent work has also identified the source of DPP-4 critical to GLP-1 and GIP degradation as being endothelial cells, rather than enteric epithelial or haematopoietic cells . Although doses of DPP-4 inhibitors that reduce enzyme activity in the intestinal lumen without prominent systemic effects (gut-selective) improve glucose tolerance and increase both GLP-1 and GIP, they are not as potent as doses that decrease plasma DPP-4 activity as well [19, 20]. Interestingly, gut-selective doses of sitagliptin retained the ability to improve glucose tolerance in mice with genetic ablation of DPP-4 from endothelial cells, albeit only in animals fed a chow diet and not those receiving a high-fat diet. The studies described here examined the acute effect of linagliptin in improving glucose tolerance in chow-fed mice and found that pancreatic GLP-1 is an important source for the inhibitory effects of DPP-4.
Our previous work in the GcgRA∆Null and GcgRA∆VilCre mouse models demonstrated that the minimal glucose excursion after an oral glucose load was not due to improved insulin sensitivity or increased insulin secretion . We hypothesise that the absence of glucagon leads to improved oral glucose tolerance similar to that seen in glucagon receptor knockout mice, which also have minimal glucose excursions after an oral glucose load. Despite this, linagliptin still improved glucose tolerance in GcgRA∆Null mice, yet we found no additivity of linagliptin and glucose on insulin levels. It is possible that we would need to assess a full time course to fully appreciate the impact of linagliptin on insulin levels in these mice. It is also possible that DPP-4 inhibitors have effects that go beyond insulin secretion. Indeed, previous work has determined that vildagliptin improves glucose tolerance secondary to increased insulin secretion and proliferation of beta cells . Overall, the key point from these data is that the ability of linagliptin to regulate glucose tolerance depends upon both GIP signalling and the tissue-specific source (i.e. pancreas) of available GLP-1.
We cannot exclude the possibility that GLP-1 or GIP receptor number or sensitivity could influence responses to ligands or antagonists of those receptors. However, in experiments where the concentration of Ex9 is doubled (data not shown), we still did not see a significant Ex9-induced impairment of glucose tolerance in GcgRA∆Null or GcgRA∆VilCre mice. As expected, linagliptin did not increase active GLP-1 levels to control levels in the GcgRA∆PDX1Cre mice as the pancreas is not a major contributor to circulating GLP-1. In line with previous reports [22, 23, 24], we believe pancreas-produced GLP-1 acts in a paracrine manner. In an important series of experiments, recent findings from a study using perifused islets demonstrated that both GLP-1 and glucagon secreted from alpha cells can act on beta cell GLP-1Rs to regulate insulin secretion . Thus, alpha cell preproglucagon products are clearly important in regulating local insulin levels (i.e. paracrine action). While DPP-4 is expressed in mouse and pig pancreatic endocrine cells [26, 27] within the pancreatic duct, and in pancreatic alpha cells in humans [28, 29], glucagon is not one of its major substrates. Thus, DPP-4 inhibitors could easily act on pancreatic islets to specifically protect the local pool of GLP-1 produced by alpha cells. Interestingly, there has been speculation that DPP-4 plays a role in mediating the balance of glucagon and GLP-1 from pancreatic alpha cells . Indeed, previous work has found that linagliptin protects human isolated islets from glucose-, lipid- and cytokine-induced toxicity . Additionally, islets isolated from individuals with type 2 diabetes exhibit decreased DPP-4 activity in order to preserve GLP-1 secretion in culture, further implicating a functional role of endogenous islet GLP-1 in diabetes .
In order to transfer preclinical knowledge about tissue-specific sources of Gcg peptides to improve upon current pharmacotherapies for type 2 diabetes, it is important to understand what stimuli provoke paracrine communication within the pancreas. Preclinical models may be used to help determine which physiological conditions regulate one pool of GLP-1 from another. Conditions such as metabolic stress, acute or chronic inflammation or nutritional availability all might impact the regulation of GLP-1 from intestinal or pancreatic sources . Indeed, it has been suggested that pancreatic Gcg-encoded peptides are essential components in adaptation to metabolic stress and that a lack of communication within the endocrine pancreas contributes to variations in glucose tolerance . Further, human islet architecture has a more favourable organisation for paracrine signalling when compared with mouse islets [22, 31]. Although we cannot rule out the possibility of species differences in GLP-1 biology, we feel that understanding the nuances of the potential importance of pancreatic Gcg-encoded peptides to islet function will be informative to future studies in humans.
Previous studies in Zucker rats suggest that GIP signalling is muted during obesity . However, we found that 11 days of vildagliptin treatment was still able to improve glucose tolerance in diet-induced obese GcgRAΔNull and GcgRAΔVilCre mice. In general, when hyperglycaemia is corrected in individuals with type 2 diabetes using a variety of interventions, including sulfonylureas and insulin, GIP sensitivity can be restored [32, 33]. Similar effects have been observed in obese rats following DPP-4 inhibitor treatment . While it is unlikely that GLP-1 and/or GIP contribute to the pathology of type 2 diabetes mellitus, pharmacotherapeutics targeting both GIP receptors and GLP-1Rs in individuals with type 2 diabetes may offer further benefits than GLP-1R agonism alone . Thus, it is possible, given the better than normal glucose tolerance inherent in the GcgRAΔNull and GcgRAΔVilCre mouse lines, that GIP signalling was preserved, therefore, enabling vildagliptin to improve glucose tolerance.
In summary, our data demonstrate that via GIP signalling linagliptin is able to retain its ability to improve glucose tolerance in mice devoid of Gcg-encoded peptides, including GLP-1 and oxyntomodulin (two targets of DPP-4 degradation). Further, using a combination of genetic and pharmacological tools, our data suggest that islet-derived rather than intestine-derived GLP-1 also contributes to the therapeutic efficacy of DPP-4 inhibitors.
We would like to thank J. Magrisso (Department of Surgery, University of Michigan, USA) for his technical assistance.
KR, JS, KL, RA and AH contributed to data acquisition, data analysis and editing of the manuscript. DD, RJS and TK were responsible for conception and experimental design, interpretation of the data and editing of the manuscript. CRH contributed to all aspects of this manuscript, including data acquisition and analysis and drafting and editing the manuscript. DAS contributed to conception, experimental design and drafting the manuscript, provided final approval of the submitted manuscript and is guarantor of this work.
This work is supported in part by NIH Awards DK107282 (DAS) and DK093848 (RJS). The investigators also received research support from Boehringer Ingelheim (DAS, RJS), Ethicon Endo-Surgery (DAS, RJS), Sanofi (RJS) and Novo Nordisk A/S (DAS, RJS). The study sponsor was not involved in the design of the study; the collection, analysis and interpretation of data; writing the report; or the decision to submit the report for publication.
Duality of interest
RJS has received research support from Ethicon Endo-Surgery, Novo Nordisk, Sanofi and Janssen. RJS has served on scientific advisory boards for Ethicon Endo-Surgery, Daiichi Sankyo, Janssen, Novartis, Nestle, Takeda, Boehringer Ingelheim, Sanofi and Novo Nordisk and is a paid speaker for Ethicon Endo-Surgery. DAS has received research support from Ethicon Endo-Surgery, Novo Nordisk and Boehringer Ingelheim. All other authors declare no duality of interest associated with this manuscript.
- 2.Nauck MA, Kind J, Köthe LDLD et al (2016) Quantification of the contribution of GLP-1 to mediating insulinotropic effects of DPP-4 inhibition with vildagliptin in healthy subjects and patients with type 2 diabetes using exendin [9-39] as a GLP-1 receptor antagonist. Diabetes 65(8):2440–2447. https://doi.org/10.2337/db16-0107 CrossRefGoogle Scholar
- 3.Meier JJ, Nauck MA, Kranz D et al (2004) Secretion, degradation, and elimination of glucagon-like peptide 1 and gastric inhibitory polypeptide in patients with chronic renal insufficiency and healthy control subjects. Diabetes 53(3):654–662. https://doi.org/10.2337/diabetes.53.3.654 CrossRefGoogle Scholar
- 7.Nauck MA, Heimesaat MM, Orskov C et al (1993) Preserved incretin activity of glucagon-like peptide 1 [7-36 amide] but not of synthetic human gastric inhibitory polypeptide in patients with type-2 diabetes mellitus. J Clin Invest 91(1):301–307. https://doi.org/10.1172/JCI116186 CrossRefGoogle Scholar
- 10.Ravn P, Madhurantakam C, Kunze S et al (2013) Structural and pharmacological characterization of novel potent and selective monoclonal antibody antagonists of glucose-dependent insulinotropic polypeptide receptor. J Biol Chem 288(27):19760–19772. https://doi.org/10.1074/jbc.M112.426288 CrossRefGoogle Scholar
- 11.Thomas L, Eckhardt M, Langkopf E et al (2008) (R)-8-(3-Amino-piperidin-1-yl)-7-but-2-ynyl-3-methyl-1-(4-methyl-quinazolin-2-ylmethyl)-3,7-dihydro-purine-2,6-dione (BI 1356), a novel xanthine-based dipeptidyl peptidase 4 inhibitor, has a superior potency and longer duration of action compared with other dipeptidyl peptidase-4 inhibitors. J Pharmacol Exp Ther 325(1):175–182. https://doi.org/10.1124/jpet.107.135723 CrossRefGoogle Scholar
- 15.Hansotia T, Baggio LL, Delmeire D et al (2004) Double incretin receptor knockout (DIRKO) mice reveal an essential role for the enteroinsular axis in transducing the glucoregulatory actions of DPP-IV inhibitors. Diabetes 53(5):1326–1335. https://doi.org/10.2337/diabetes.53.5.1326 CrossRefGoogle Scholar
- 16.Flock G, Baggio LL, Longuet C, Drucker DJ (2007) Incretin receptors for glucagon-like peptide 1 and glucose-dependent insulinotropic polypeptide are essential for the sustained metabolic actions of vildagliptin in mice. Diabetes 56(12):3006–3013. https://doi.org/10.2337/db07-0697 CrossRefGoogle Scholar
- 33.Højberg PV, Vilsbøll T, Rabøl R et al (2009) Four weeks of near-normalisation of blood glucose improves the insulin response to glucagon-like peptide-1 and glucose-dependent insulinotropic polypeptide in patients with type 2 diabetes. Diabetologia 52(2):199–207. https://doi.org/10.1007/s00125-008-1195-5 CrossRefGoogle Scholar
- 34.Frias JP, Nauck MA, Van J et al (2018) Efficacy and safety of LY3298176, a novel dual GIP and GLP-1 receptor agonist, in patients with type 2 diabetes: a randomised, placebo-controlled and active comparator-controlled phase 2 trial. Lancet 392(10160):2180–2193. https://doi.org/10.1016/S0140-6736(18)32260-8 CrossRefGoogle Scholar