Dipeptidyl peptidase 4 (DPP-4) is expressed in mouse and human islets and its activity is decreased in human islets from individuals with type 2 diabetes
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Inhibition of the enzyme dipeptidyl peptidase 4 (DPP-4), which cleaves and inactivates glucagon-like peptide 1 (GLP-1), is a glucose-lowering strategy in type 2 diabetes. Since DPP-4 is a ubiquitously distributed enzyme, we examined whether it is expressed in islets and whether an islet effect to inhibit DPP-4 may result in stimulated insulin secretion.
We investigated DPP-4 expression and activity in the islets of mouse models of obesity as well as human islets from non-diabetic and type 2 diabetic donors. We further investigated whether inhibition with DPP-4 inhibitors could promote insulin secretion via islet GLP-1 in isolated islets.
DPP-4 was readily detected in mouse and human islets with species-specific cellular localisation. In mice, DPP-4 was expressed predominantly in beta cells, whereas in humans it was expressed nearly exclusively in alpha cells. DPP-4 activity was significantly increased in islets from diet-induced obese mice compared with mice fed a control diet. In humans, DPP-4 activity was significantly lower in islets from type 2 diabetic donors than in non-diabetic donors. In human islets, there was a significant positive correlation between DPP-4 activity and insulin secretory response to 16.7 mmol/l glucose. Treatment of mouse islets with the DPP-4 inhibitors, NVPDPP728 and vildagliptin, resulted in a significant potentiation of insulin secretion in a GLP-1-dependent manner, as this was inhibited by the GLP-1 receptor antagonist, Exendin (9-39), and was retained in glucose-dependent insulinotropic polypeptide (GIP) receptor-deficient mice but lost in mice lacking GLP-1 receptors or both incretin receptors. Human islets treated with the DPP-4 inhibitor, vildagliptin, showed increased secretion of insulin and intact GLP-1.
We conclude that DPP-4 is present and active in mouse and human islets, is regulated by the disease state, and that inhibition of islet DPP-4 activity can have direct effects on islet function. Inhibiting islet DPP-4 activity may therefore contribute to the insulin-secretory and glucose-lowering action of DPP-4 inhibition.
KeywordsGLP-1 Insulin secretion Islets of Langerhans Obesity Type 2 diabetes
Double incretin receptor knockout
Dipeptidyl peptidase 4
Glucose-dependent insulinotropic polypeptide
Glucagon-like peptide 1
Normal chow diet
Prohormone convertase 1/3
Stromal cell-derived factor-1
The incretin hormones, glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide 1 (GLP-1), are insulinotropic factors secreted from the intestine in response to nutrient intake . The intact, active form of the incretin hormones are short-lived peptides with half-lives of 2–7 min in humans . The short half-life of the incretins is due to their rapid degradation by the serine protease, dipeptidyl peptidase 4 (DPP-4) . DPP-4 is abundantly expressed by vascular endothelial cells and is present in the circulation in both membrane-bound and soluble forms . Inhibition of DPP-4 results in higher circulating levels of intact GLP-1 and GIP and increased insulin secretion . Consequently, numerous pharmacological DPP-4 inhibitors are being used in clinical practice for the treatment of type 2 diabetes .
Recent studies have suggested that DPP-4 inhibition exerts local glucose-lowering effects that are independent of inhibition of circulating DPP-4 activity. Thus, oral administration of a low dose of DPP-4 inhibitor to mice reduced glucose without inhibiting the circulating DPP-4 activity [7, 8]. This would suggest that local DPP-4 activity is also important for the glucose-lowering action of DPP-4 inhibitors. In addition, numerous studies have demonstrated that GLP-1 is secreted, not only in the intestine, but also in the pancreatic islets due to alternative processing of proglucagon [9, 10, 11, 12, 13]. Regulation of islet GLP-1 by glucose, cytokines, chemokines and the diabetic state has been demonstrated, and it has been shown to be increased in islets of type 2 diabetic humans [11, 12, 13, 14]. This would make it possible that DPP-4 inhibitors may also work through a direct islet action, provided that islets express DPP-4 with DPP-4 activity. However, the regulation of islet GLP-1 by DPP-4 has not been investigated to date. This study therefore examined the expression, cellular localisation, activity and functional consequences of DPP-4 in islets from multiple mouse models and healthy and type 2 diabetic human donors.
Human pancreatic biopsy samples were obtained from five non-diabetic donors and fixed in formaldehyde before paraffin embedding and tissue sectioning, with approval from the local ethics committee in Pisa. Human islets were obtained from cadaver donors courtesy of the Nordic Network for Clinical Islet Transplantation (www.nordicislets.org; accessed 1 March 2014), Uppsala University, with appropriate ethics permission obtained from the regional ethics committees in Lund and Uppsala.
Six-week-old male and female C57BL6/JBomTac mice were purchased from Taconic Europe (Skensved, Denmark). For studies of diet-induced obesity, mice were fed ad libitum with either normal control chow (ND) or a high-fat diet (HFD) (D12492 Research Diets, New Brunswick, NJ, USA) containing 60% kJ from fat. Glucagon receptor-deficient mice (Gcgr −/−) were derived as previously described . Double incretin receptor knockout (DIRKO) mice were derived as previously described . Single GIP receptor- and GLP-1 receptor-deficient mice were obtained by crossing wild-type C57BL6 mice with DIRKO mice and pairing the subsequent heterozygotes.
Human pancreatic sections were obtained as described above. Formaldehyde-fixed, paraffin-embedded mouse pancreatic sections (5 μm thick) were obtained from mice fed with ND or HFD (for 16 weeks) as well as Gcgr −/− mice. After dewaxing of the sections, the antibody-binding epitopes of DPP-4 were retrieved by heating in a water bath at 95°C for 20 min in sodium citrate buffer (10 mmol/l sodium citrate, 0.05% Tween 20, pH 6.0). Sections were incubated overnight at 4°C with primary antibodies against mouse DPP-4 (polyclonal goat anti-mouse DPP-4/CD26 antibody; R&D Systems, Minneapolis, MN, USA), human DPP-4 (polyclonal goat anti-human DPP-4 antibody; R&D Systems), insulin (polyclonal guinea pig anti-insulin antibody; EuroProxima, Arnhem, the Netherlands), glucagon (monoclonal mouse anti-glucagon antibody; Abcam, Cambridge, UK), amidated GLP-1 (monoclonal mouse anti-GLP-17–36amide (8G9); Enzo Life Sciences, Farmingdale, NY, USA) and somatostatin (polyclonal rabbit anti-somatostatin antibody; a gift from Dr Eva Ekblad, Lund University, Lund, Sweden, originally from Dr Jens J. Holst, University of Copenhagen, Copenhagen Denmark) at 1:200, 1:200, 1:2,000, 1:500 and 1:3,000 dilution, respectively. Specificity of the DPP-4 antibody was validated by preblocking with human CD26 peptide (Abcam). The fluorescent secondary antibodies used were: polyclonal donkey anti-goat (Alexa Fluor 488 conjugated; Molecular Probes, Eugene, OR, USA) for mice/human DPP-4; polyclonal rabbit anti-guinea pig (DyLight 594 conjugated; Abcam) for insulin; polyclonal donkey anti-mouse (Alexa Fluor 594 conjugated; Abcam) for glucagon; and polyclonal goat anti-rabbit (Alexa Fluor 546 conjugated; Abcam) for somatostatin. All of the secondary antibodies were diluted 1:200. Fluorescence images were captured with a Carl Zeiss LSM 780 confocal microscope with Zen imaging software.
Isolation of mouse and human islets
Human islets were isolated from pancreases from non-diabetic and type 2 diabetic organ donors by collagenase digestion as previously described  and cultured in CMRL 1066 medium (ICN Biomedicals, Costa Mesa, CA, USA) supplemented with 10 mmol/l HEPES, 2 mmol/l l-glutamine, 50 μg/ml gentamicin, 0.25 μg/ml Fungizone (Gibco, Gaithersburg, MD, USA), 20 μg/ml ciprofloxacin (Bayer Healthcare, Leverkusen, Germany) and 10 mmol/l nicotinamide at 37°C (5% CO2) for 1–5 days before handpicking and the determination of DPP-4 activity. Mouse islets were isolated by collagenase digestion and handpicked in an inverted microscope. Batches of freshly isolated islets were preincubated in HEPES balanced salt solution containing 125 mmol/l MgCl2, 25 mmol/l HEPES (pH 7.4), 5.6 mmol/l glucose and 0.1% fatty acid-free BSA (Boehringer Mannheim, Mannheim, Germany) for 60 min before insulin secretion experiments.
Analysis of gene expression
Gene expression analysis was carried out by microarray on cDNA from islets of 23 type 2 diabetic donors and 23 non-diabetic donors matched for age, sex and BMI, using the Affymetrix GeneChip Human Gene 1.0 ST whole-transcript assay as previously described [18, 19]. The data were summarised and normalised with the robust multi-array analysis method using the Expression Console software package (Affymetrix, Santa Clara, CA, USA). The full dataset is available on the Entrez GEO database with accession number GSE54279.
Glucose-stimulated insulin secretion
Insulin secretion in human islets was determined with islet perifusion and static islet incubation. Shortly after isolation from human donors, islets were subjected to glucose perifusion experiments as previously described . Briefly, 20 islets were perifused with low (1.67 mmol/l) glucose for 40 min before perifusion with high (16.7 mmol/l) glucose for 40 min and returning to baseline 1.67 mmol/l glucose perifusion. The stimulatory index was calculated as the ratio of insulin released during high-glucose perifusion to the insulin released during low-glucose perifusion . For static incubations, human or mouse islets were isolated as described above, then incubated in groups of three in 200 μl of the buffer described above with 2.8 or 16.7 mmol/l glucose in the presence or absence of 100 or 1,000 nmol/l NVPDPP728 (Tocris Biosciences, Bristol, UK) or 100, 500 or 1,000 nmol/l vildagliptin (gift from Novartis Pharmaceutical, East Hannover, NJ, USA).
Intact GLP-1 secretion from human islets
Human islets were obtained as described above and incubated in batches of 40 islets per condition for 24 h. After the incubations, the supernatant fractions were removed, and the DPP-4 inhibitor, diprotin A, was added to prevent degradation of the intact peptide.
Assay of DPP-4 activity
DPP-4 activity in islets and incubation media was determined as previously described with Gly-Pro p-nitroanilide as the substrate . Briefly, islets were isolated as above and disrupted by sonication in 100 μl buffer containing 50 mmol/l N-[tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid (TES), 2 mmol/l EGTA, 1 mmol/l EDTA, 250 mmol/l sucrose and 40 mmol/l phenyl phosphate, pH 7.4. Then 20 μl sonicate or medium was incubated with 100 μl 1 mmol/l Gly-Pro p-nitroanilide for 60 min at 37°C. The amount of p-nitroaniline liberated during 60 min was compared with a 1 mmol/l nitroaniline standard.
Insulin secretion was determined with mouse and human ELISA (Mercodia, Uppsala, Sweden). Human intact GLP-1 (7-36 amide and 7-37) was determined by an ELISA that has no detectable cross reactivity with glucagon and detects intact GLP-1 in mammalian species (Millipore, Billerica, MA, USA).
Data are presented as mean ± SEM unless otherwise indicated. Correlation analysis was performed with Spearman’s correlation test. For mouse islet experiments, differences between groups were determined using Student’s unpaired t test. For human islet experiments, differences between treatment groups were determined using paired t test. For all analyses, statistical significance was defined as p < 0.05. Analyses were carried out using the Prism 6.0 software package (Graphpad, San Diego, CA, USA).
DPP-4 is expressed in human and mouse islets in a cell- and species-specific manner
Islet DPP4 gene expression does not differ between type 2 diabetic and non-diabetic donors
Islet DPP-4 activity is increased in obese mice and decreased in type 2 diabetic humans
Inhibition of DPP-4 activity in islets results in increased insulin and intact GLP-1 secretion
We describe the expression and activity of DPP-4 in mouse and human islets under conditions of obesity and type 2 diabetes. DPP-4 was expressed in islets of both species and in all models examined. Although DPP-4 activity in islets was lower than in plasma, it was clearly regulated in response to different metabolic conditions. In islets of obese mice chronically fed an HFD, DPP-4 activity was increased. In contrast, in human islets from type 2 diabetic donors, DPP-4 activity was decreased. In fact, there was a positive relationship between insulin secretion and DPP-4 activity in the human islets, and a trend in mouse islets, suggesting that insulin may play a role in regulating DPP-4 activity in human islets. Inhibition of DPP-4 in mouse and human islets resulted in a direct enhancement of insulin secretion itself in this study. Thus, DPP-4 is not only present and active in mouse and human islets, but inhibition of islet DPP-4 activity also has a direct stimulatory effect on insulin secretion, which is GLP-1 dependent.
The first demonstration of DPP-4 expression in islets was in pigs, showing exclusive expression in the alpha cells [25, 26]. To our knowledge the protein localisation of DPP-4 has not previously been shown in mouse or human islets. Mouse islets showed a near-exclusive expression of DPP-4 in beta cells, with little expression in alpha cells. Human islets were the opposite and, like pig islets, expressed DPP-4 almost exclusively in alpha cells. The species difference in the localisation of DPP-4 expression, and the possible physiological consequence of that difference, is unclear. Rodent and human islets differ somewhat in the distribution of islet cells, as well as in their vascularisation and innervation . The species specificity of DPP-4 cellular localisation requires further investigation in other species and will be addressed in future studies.
In both the mouse and human pancreatic sections, there was a granular distribution of DPP-4 staining, suggesting that DPP-4 is secreted into the intracellular space as suggested in the earlier pig studies . Shah and colleagues recently demonstrated that DPP-4 activity was detectable in the media of human islet incubations and that this activity was inhibited by the DPP-4 inhibitor, linagliptin, suggesting that it is secreted from human islets as well .
Previous studies have suggested that locally produced and secreted GLP-1 from rodent and human islets exerts beneficial effects on beta cell development, function and protection [28, 29]. Some of these studies demonstrated increased islet GLP-1 in diabetic states and suggested increased prohormone convertase 1/3 (PC1/3) expression as the primary regulator of islet GLP-1 [10, 11, 13]. We confirm that GLP-1 is present in the islets in our study and demonstrate this for the first time in diet-induced obese mice and glucagon receptor knockout mice. While GLP-1 will not be produced in alpha cells in appreciable amounts without increased PC1/3 expression and activity, the functional effects of GLP-1 are dependent on the peptide reaching its receptor in its intact form. The tight spatial relationship between alpha and beta cells means that the intact GLP-1 secreted from alpha cells should reach the adjacent beta cells and act in a paracrine manner. This would be the case unless DPP-4 was secreted into the space between alpha and beta cells. Hence, the local production of GLP-1 and DPP-4 may be tightly regulated and of relevance for islet function.
Interestingly, we demonstrate that human islets from type 2 diabetic patients have decreased DPP-4 activity. This corresponded to higher secretion of intact GLP-1 from the islets in overnight culture. Not only does the islet respond to the diabetic milieu by increasing PC1/3 expression, and thereby GLP-1 production, it also concurrently decreases DPP-4 activity to preserve the GLP-1 that is being produced in larger amounts. Therefore decreased DPP-4 activity and increased PC1/3 expression may both contribute to the increased secretion of intact GLP-1 seen in type 2 diabetic human islets. This implies that endogenous islet GLP-1 may play a functional role in diabetes.
Liu et al demonstrated that the chemokine, stromal cell-derived factor-1 (SDF-1), increases GLP-1 production from islets . SDF-1 was shown to be produced by beta cells and is itself a substrate for DPP-4, as demonstrated in vitro and in vivo . In this case, a decrease in DPP-4 activity within the islet would result in more intact forms of SDF-1, which would in turn help drive further GLP-1 production from the islet. Other substrates of DPP-4 that have been shown to be expressed in islets include GIP, interferon γ inducible protein 10 (IP-10) and pituitary adenylate cyclase-activating polypeptide (PACAP) [31, 32, 33]. It remains to be determined what effect the decrease in DPP-4 activity seen in islets from type 2 diabetic patients has on the resulting action of these substrates.
Inhibitors of DPP-4 are now established as effective treatments for type 2 diabetes . Our results suggest that DPP-4 inhibitors can exert direct, GLP-1-dependent, effects on target tissues independent of plasma DPP-4 activity. This has been previously demonstrated in vivo by Waget and colleagues  using the DPP-4 inhibitor, sitagliptin, confirmed by our group with vildagliptin , and shown in vitro in human islets using the DPP-4 inhibitor, linagliptin . Human islets from non-diabetic donors that had been incubated under hyperglycaemic–hyperlipidaemic conditions showed increased secretion of insulin and intact GLP-1, as well as reduced beta cell apoptosis, when treated with linagliptin . There are numerous DPP-4 inhibitors used in clinical practice, and, although they all function to inhibit the peptidase activity of DPP-4, they are not all the same in their pharmacokinetic and pharmacodynamic profiles. Linagliptin and vildagliptin differ in their pharmacokinetic and pharmacodynamic properties. Vildagliptin forms covalent bonds with DPP-4 and is itself metabolised by DPP-4 , while linagliptin is a competitive DPP-4 inhibitor which does not covalently bond with DPP-4. With these differences, it stands to reason that different DPP-4 inhibitors have different tissue-specific effects.
An intriguing finding of this study is the positive correlation between DPP-4 activity in the islet and insulin secretion. It is logical that, if production of GLP-1 within alpha cells increases in islets of type 2 diabetic individuals, as has been demonstrated , a decrease in islet DPP-4 activity would result in more intact GLP-1 secreted from the alpha cell being able to act on its receptor. The correlation suggests that DPP-4 activity declines as beta cell function declines. One could speculate that insulin provides some measure of regulation of DPP-4 activity and, as tonic insulin secretion decreases, DPP-4 activity decreases with it. Conversely, in insulin-resistant individuals who adequately compensate by increasing insulin secretion, increased insulin action on the beta cell may increase DPP-4 activity. The regulation of DPP-4 by insulin and the change in DPP-4 activity over the natural history of diabetes will need to be determined. This is the subject of ongoing studies.
In summary, DPP-4 is present in isolated islets from mice and humans, and its activity changes under different pathophysiological conditions. DPP-4 inhibitors exert direct effects on secretion of insulin and intact GLP-1 and thus may promote islet function in part by acting directly on islet DPP-4.
The authors acknowledge K. Andersson, M. Anderberg and L. Ohlsson of the Department of Clinical Sciences, Lund University, Lund, Sweden for technical assistance, and U. Krus and Å. Nilsson of the Lund University Diabetes Center Human Tissue Laboratory, Malmö, Sweden and the Nordic Network for Clinical Islet Transplantation, Uppsala University, Uppsala, Sweden for provision of the human islets and associated information. The authors thank E. Zhang of the Department of Clinical Sciences, Lund University, Malmö, Sweden for assistance with the confocal microscopy. The authors also acknowledge L. Marselli of the Department of Endocrinology and Metabolism, University of Pisa, Pisa, Italy for the provision of the human pancreatic tissue sections.
This work was supported by grants from the Swedish Research Council, Lund University Medical Faculty, ALF/Region Skåne, Knut and Alice Wallenberg Foundation (confocal microscope) and the Royal Physiographical Society of Lund.
Duality of interest
BA is a member of Novartis’ speaker’s bureau. All other authors declare that there is no duality of interest associated with their contribution to this manuscript.
LL designed and performed experiments, analysed data, wrote the manuscript and approved the final version. YY and YS provided experimental material and conceptual design, revised the manuscript and approved the final version. PM provided conceptual design and experimental material and revised and approved the final version of manuscript. BAO designed the study, performed experiments, analysed data, wrote the manuscript and approved the final version. BA designed the study, analysed data, wrote and revised the manuscript and approved the final version, and is the guarantor of this work.
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