Clinical and pathological characterization of a novel transgenic animal model of diabetes mellitus expressing a dominant negative glucose-dependent insulinotropic polypeptide receptor (GIPR dn)

Clinical and pathological characterization of a novel transgenic animal model of diabetes mellitus expressing a dominant negative glucose-dependent insulinotropic polypeptide receptor (GIPR dn) Gastrointestinal hormones like glucose-dependent insulinotropic polypeptide have recently been shown to be involved in the pathogenesis of diabetes mellitus in humans and animals models of diabetes mellitus. The aim of this study was to characterize a novel transgenic mouse model expressing a dominant negative glucosedependent insulinotropic polypeptide receptor (GIPRdn) under the control of the rat insulin gene promoter and their non-transgenic counterparts. Detailed analysis of clinical parameters was performed, including urine glucose, blood or serum glucose and serum insulin values. In addition, oral and subcutaneous glucose tolerance tests were performed, and HbA1c levels and various serum parameters were determined. The detection of the daily food and water intake and the daily urine volume was performed in two age groups. Further, body and organ weights were determined. Qualitative and quantitative morphological changes of the pancreas and the kidneys were investigated in several age groups. Some of the parameters were studied in different diet groups, one of them received standard rodent chow, and the other received a carbohydrate-restricted diet until four months of age. All transgenic mice studied exhibited severe glucosuria from 21 days of age onwards. From 30 days of age onwards, GIPRdn transgenic males and females showed severe hyperglycemia and hypoinsulinemia (p<0.05). In male transgenic animals, the fasted body weight was found to be lower than in age-matched male control mice. The daily food and water intake and the 24-hour urine volume were significantly higher in all transgenic animals investigated. Histological and immunohistochemical survey of the pancreas revealed a striking change of the islet cell composition and distribution. Further, quantitative- stereological analysis of the pancreas revealed a significant reduction of the total volumes of pancreatic islets, B-cells in the islets and isolated B-cells in the exocrine pancreas indicating neogenesis of islets. Kidney changes included renal and glomerular hypertrophy, glomerulosclerosis and tubulo-interstitial changes. In conclusion, transgenic mice expressing a dominant negative GIP receptor under the control of the rat insulin gene promoter develop a severe diabetic phenotype and striking 138 histological changes of the endocrine pancreas. Further, advanced diabetesassociated organ lesions, particularly of the kidney were observed and therefore, GIPRdn transgenic mice are considered a valuable model for studying long-term complications of diabetes mellitus.Klinische und pathomorphologische Befunde bei einem neuartigen transgenen diabetischen Tiermodell, das einen dominant negativen glucose-dependent insulinotropic polypeptide Rezeptor (GIPR dn) exprimiert. In den letzten Jahren wurde beim Studium der Pathogenese des Diabetes mellitus die Aufmerksamkeit zunehmend auf die Beteiligung gastrointestinaler Hormone wie zum Beispiel glucose-dependent insulinotropic polypeptide gerichtet. Ziel dieser Studie war es, bei einem neuartigen transgenen Tiermodell, klinische und pathomorphologische Veränderungen eingehend zu charakterisieren. Bei den untersuchten Tieren handelte es sich um transgene Mäuse, die einen dominant negativen glucosedependent insulinotropic polypeptide Rezeptor (GIPRdn) unter der Kontrolle des Ratteninsulingenpromotors exprimieren und um nicht-transgene Geschwistertiere. Es erfolgte eine detaillierte Untersuchung Diabetes-relevanter klinischer Parameter, unter der Berücksichtigung von Harnglukoseausscheidung, Blut- bzw. Serumglukose- und Seruminsulinwerten. Zusätzlich wurden orale und subkutane Glukosetoleranztests durchgeführt und HbA1c -Werte sowie verschiedene Serumparameter bestimmt. Die tägliche Futter- und Wasseraufnahme und das tägliche Harnvolumen wurden bei zwei Altersgruppen gemessen. Körper- und Organgewichte wurden ebenfalls erfasst. Die Erfassung morphologischer und histopathologischer Veränderungen des Pankreas und der Nieren erfolgte sowohl qualitativ als auch quantitativ-stereologisch an mehreren Altersgruppen. Einige der untersuchten Parameter wurden an Tieren, die mit Haltungsfutter gefüttert wurden und an Vergleichstieren erhoben, die bis zum Alter von 4 Monaten mit einer kohlenhydratarmen Diät ernährt wurden. Alle untersuchten transgenen Tiere zeigten ab einem Alter von 21 Tagen eine schwere Glukosurie. Im Alter von 30 Tagen zeigten GIPRdn transgene männliche und weibliche Mäuse eine hochgradige Hyperglykämie und eine hochgradige Hypoinsulinämie. Bei männlich- transgenen Tieren war das Köpergewicht im Vergleich zu gleichgeschlechtlichen 139 Kontrolltieren reduziert (p<0.05). Die tägliche Futter- und Wasseraufnahme und das tägliche Harnvolumen war bei allen untersuchten transgenen Tieren signifikant höher als bei Kontrolltieren. Histologische und immunhistochemische Untersuchungen am Pankreas zeigten schwere Veränderungen der Zusammensetzung und Verteilung der Inselzellen auf. Diese qualitativen Befunde konnten durch quantitativstereologische Untersuchungen eingehend charakterisiert werden. Das Gesamtvolumen der Pankreasinseln und der B-Zellen in den Inseln war bei GIPRdn transgenen Mäusen signifikant niedriger als bei Kontrolltieren. Gleiches gilt für das Gesamtvolumen isolierter B-Zellen im exokrinen Pankreas, die als Indikator für Inselneogenese angesehen werden. Die festgestellten Nierenveränderungen umfassen renale und glomeruläre Hypertrophie, Glomerulosklerose und tubulointerstitielle Veränderungen. Die quantitativ-stereologische Auswertung konnte die subjektiven Befunde bestätigen, sowohl das Nierenvolumen als auch das mittlere Glomerulumvolumen waren bei transgenen Mäusen signifikant erhöht. Aus den erhobenen Befunden ergibt sich die Schlussfolgerung, dass transgene Mäuse, die einen dominant-negativen GIP Rezeptor unter der Kontrolle des Ratteninsulingenpromoters exprimieren eine hochgradigen diabetischen Phänotyp und prägnante histologische Veränderungen am endokrinen Pankreas entwickeln. Weiterhin konnten Diabetes-assoziierte Alterationen diverser Organe, insbesondere der Niere festgestellt werden, woraufhin GIPRdn transgene Mäuse als ein wertvolles Tiermodell für die Untersuchung diabetischer Spätkomplikationen angesehen werden

Multiple Glucagon-Producing Pancreatic Neuroendocrine Tumors in a Horse (Equus caballus)

Pancreatic neuroendocrine tumors of glucagon-producing cells are extremely rare in domestic animals. In this report, we describe for the first time, to our knowledge, the incidental finding of multiple glucagon-producing neuroendocrine tumors of the pancreas of a horse. The animal was euthanized due to severe local infection after tooth extraction. On postmortem examination, multiple white nodules of up to 4 cm in diameter were observed in the pancreas. Histologically, pancreatic nodules had the appearance of neuroendocrine neoplasms with positive immunoreactivity for glucagon, synaptophysin, chromogranin A, and neuron-specific enolase. Electron microscopy revealed numerous electron-dense granules, similar to those observed in normal pancreatic alpha cells, in the neoplastic cells. In addition, the left adrenal gland showed multiple hyperplastic foci and adenomas in the medulla that were identified as pheochromocytomas. Based on the morphologic appearance and immunohistochemical staining pattern of pancreatic nodules, a diagnosis of multiple glucagon-producing neuroendocrine tumors was made

Gene expression profiling of bovine peripartal placentomes: detection of molecular pathways potentially involved in the release of foetal membranes

The mechanisms underlying detachment of foetal membranes after birth in cows are still unclear. To address this problem in a systematic manner, we performed the first holistic transcriptome study of bovine placentomes antepartum (AP; n=4 cows) and intrapartum (IP; n=4 cows) using Affymetrix GeneChip Bovine Genome Arrays. Three placentomes were extracted from each cow, and tissue samples from the contact zones of the placentomes (foeto-maternal units) were recovered by systematic random sampling and processed for RNA extraction and for stereological quantification of cellular composition. Statistical analysis of microarray data (false discovery rate 1%) revealed 759 mRNAs with at least twofold higher levels in the samples of the AP group, whereas 514 mRNAs showed higher levels in the IP group. The differentially expressed genes were classified according to biological processes and molecular functions using the Functional Annotation Clustering tool of the DAVID Bioinformatics Resources. Genes with higher mRNA levels in the AP group were nearly completely related to mitotic cell cycle and tissue differentiation. During parturition, a complete shift occurred because the genes with higher mRNA levels in IP were nearly all related to three different physiological processes/complexes: i) apoptosis, ii) degradation of extra cellular matrix and iii) innate immune response, which play a fundamental role in placental detachment. These results are an excellent basis for future studies investigating the molecular basis of retained foetal membranes

A mouse model for ulcerative colitis based on NOD-scid IL2R gamma(null) mice reconstituted with peripheral blood mononuclear cells from affected individuals

Animal models reflective of ulcerative colitis (UC) remain a major challenge, and yet are crucial to understand mechanisms underlying the onset of disease and inflammatory characteristics of relapses and remission. Mouse models in which colitis-like symptoms are induced through challenge with toxins such as oxazolone, dextran sodium sulfate (DSS) or 2,4,6-trinitrobenzenesulfonic acid (TNBS) have been instrumental in understanding the inflammatory processes of UC. However, these neither reflect the heterogeneous symptoms observed in theUC-affected population nor can they be used to test the efficacyof inhibitors developed against human targets where high sequence and structural similarity of the respective ligands is lacking. In an attempt to overcome these problems, we have developed a mouse model that relies on NOD-scid IL2R gamma(null) mice reconstituted with peripheral blood mononuclear cells derived from UC-affected individuals. Upon challenge with ethanol, mice developed colitis-like symptoms and changes in the colon architecture, characterized by influx of inflammatory cells, edema, crypt loss, crypt abscesses and epithelial hyperplasia, as previously observed in immune-competent mice. TARC, TGF beta 1 and HGF expression increased in distal parts of the colon. Analysis of human leucocytes isolated from mouse spleen revealed an increase in frequencies of CD1a+, CD64+, CD163+ and TSLPR+ CD14+ monocytes, and antigen-experienced CD44+ CD4+ andCD8+ T-cells in response to ethanol. Analysis of human leucocytes from the colon of challenged mice identified CD14+ monocytes and CD11b+ monocytes as the predominant populations. Quantitative real-time PCR (RT-PCR) analysis from distal parts of the colon indicated that IFN gamma might be one of the cytokines driving inflammation. Treatment with infliximab ameliorated symptoms and pathological manifestations, whereas pitrakinra had no therapeutic benefit. Thus, this model is partially reflective of the human disease and might help to increase the translation of animal and clinical studies

Gene expression profiling of bovine peripartal placentomes: detection of molecular pathways potentially involved in the release of foetal membranes

The mechanisms underlying detachment of foetal membranes after birth in cows are still unclear. To address this problem in a systematic manner, we performed the first holistic transcriptome study of bovine placentomes antepartum (AP; n=4 cows) and intrapartum (IP; n=4 cows) using Affymetrix GeneChip Bovine Genome Arrays. Three placentomes were extracted from each cow, and tissue samples from the contact zones of the placentomes (foeto-maternal units) were recovered by systematic random sampling and processed for RNA extraction and for stereological quantification of cellular composition. Statistical analysis of microarray data (false discovery rate 1%) revealed 759 mRNAs with at least twofold higher levels in the samples of the AP group, whereas 514 mRNAs showed higher levels in the IP group. The differentially expressed genes were classified according to biological processes and molecular functions using the Functional Annotation Clustering tool of the DAVID Bioinformatics Resources. Genes with higher mRNA levels in the AP group were nearly completely related to mitotic cell cycle and tissue differentiation. During parturition, a complete shift occurred because the genes with higher mRNA levels in IP were nearly all related to three different physiological processes/complexes: i) apoptosis, ii) degradation of extra cellular matrix and iii) innate immune response, which play a fundamental role in placental detachment. These results are an excellent basis for future studies investigating the molecular basis of retained foetal membranes

Attenuation of cardiac hypertrophy by G-CSF is associated with enhanced migration of bone marrow-derived cells

Granulocyte-colony stimulating factor (G-CSF) has been shown to promote mobilization of bone marrow-derived stem cells (BMCs) into the bloodstream associated with improved survival and cardiac function after myocardial infarction. Therefore, the aim of the present study was to investigate whether G-CSF is able to attenuate cardiac remodelling in a mouse model of pressure-induced LV hypertrophy focusing on mobilization and migration of BMCs. LV hypertrophy was induced by transverse aortic constriction (TAC) in C57BL/6J mice. Fourweeks after TAC procedure. Mice were treated with G-CSF (100g/kg/day;Amgen Biologicals) for 2weeks. The number of migrated BMCs in the heart was analysed by flow cytometry. mRNA expression and protein level of different growth factors in the myocardium were investigated by RT-PCR and ELISA. Functional analyses assessed by echocardiography and immunohistochemical analysis were performed 8weeks after TAC procedure. G-CSF-treated animals revealed enhanced homing of VLA-4(+) and c-kit(+) BMCs associated with increased mRNA expression and protein level of the corresponding homing factors Vascular cell adhesion protein 1 and Stem cell factor in the hypertrophic myocardium. Functionally, G-CSF significantly preserved LV function after TAC procedure, which was associated with a significantly reduced area of fibrosis compared to control animals. Furthermore, G-CSF-treated animals revealed a significant improvement of survival after TAC procedure. In summary, G-CSF treatment preserves cardiac function and is able to diminish cardiac fibrosis after induction of LV hypertrophy associated with increased homing of VLA-4(+) and c-kit(+) BMCs and enhanced expression of their respective homing factors VCAM-1 and SCF

Effects of the glucagon-like peptide-1 receptor agonist liraglutide in juvenile transgenic pigs modeling a pre-diabetic condition

Background: The glucagon-like peptide-1 receptor (GLP1R) agonist liraglutide improves glycemic control and reduces body weight of adult type 2 diabetic patients. However, efficacy and safety of liraglutide in adolescents has not been systematically investigated. Furthermore, possible pro-proliferative effects of GLP1R agonists on the endocrine and exocrine pancreas need to be further evaluated. We studied effects of liraglutide in adolescent pigs expressing a dominant-negative glucose-dependent insulinotropic polypeptide receptor (GIPRdn) in the beta-cells, leading to a pre-diabetic condition including disturbed glucose tolerance, reduced insulin secretion and progressive reduction of functional beta-cell mass. Methods: Two-month-old GIPRdn transgenic pigs were treated daily with liraglutide (0.6-1.2 mg per day) or placebo for 90 days. Glucose homeostasis was evaluated prior to and at the end of the treatment period by performing mixed meal and intravenous glucose tolerance tests (MMGTT and IVGTT). Finally animals were subjected to necropsy and quantitative-stereological analyses were performed for evaluation of alpha-and beta-cell mass, beta-cell proliferation as well as acinus-cell proliferation. Results: MMGTT at the end of the study revealed 23% smaller area under the curve (AUC) for glucose, a 36% smaller AUC insulin, and improved insulin sensitivity, while IVGTT showed a 15% smaller AUC glucose but unchanged AUC insulin in liraglutide-vs. placebo-treated animals. Liraglutide led to marked reductions in body weight gain (-31%) and food intake (-30%) compared to placebo treatment, associated with reduced phosphorylation of insulin receptor beta (INSRB)/insulin-like growth factor-1 receptor beta (IGF1RB) and protein kinase B (AKT) in skeletal muscle. Absolute alpha-and beta-cell mass was reduced in liraglutide-treated animals, but alpha-and beta-cell mass-to-body weight ratios were unchanged. Liraglutide neither stimulated beta-cell proliferation in the endocrine pancreas nor acinus-cell proliferation in the exocrine pancreas, excluding both beneficial and detrimental effects on the pig pancreas. Conclusions: Although plasma liraglutide levels of adolescent transgenic pigs treated in our study were higher compared to human trials, pro-proliferative effects on the endocrine or exocrine pancreas or other liraglutide-related side-effects were not observed

Synergy between CD26/DPP-IV Inhibition and G-CSF Improves Cardiac Function after Acute Myocardial Infarction

SummaryIschemic cardiomyopathy is one of the main causes of death, which may be prevented by stem cell-based therapies. SDF-1α is the major chemokine attracting stem cells to the heart. Since SDF-1α is cleaved and inactivated by CD26/dipeptidylpeptidase IV (DPP-IV), we established a therapeutic concept—applicable to ischemic disorders in general—by combining genetic and pharmacologic inhibition of DPP-IV with G-CSF-mediated stem cell mobilization after myocardial infarction in mice. This approach leads to (1) decreased myocardial DPP-IV activity, (2) increased myocardial homing of circulating CXCR-4+ stem cells, (3) reduced cardiac remodeling, and (4) improved heart function and survival. Indeed, CD26 depletion promoted posttranslational stabilization of active SDF-1α in heart lysates and preserved the cardiac SDF-1-CXCR4 homing axis. Therefore, we propose pharmacological DPP-IV inhibition and G-CSF-based stem cell mobilization as a therapeutic concept for future stem cell trials after myocardial infarction

Postnatal Development of Numbers and Mean Sizes of Pancreatic Islets and Beta-Cells in Healthy Mice and GIPRdn 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 GIPRdn transgenic mice, which exhibit an early reduction of beta-cell mass. Pancreata of female control and GIPRdn 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 GIPRdn 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 GIPRdn transgenic mice, and serum insulin to pancreatic insulin content in response to GLP-1 and arginine was significantly higher in GIPRdn 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. GIPRdn transgenic mice show a disturbed expansion of the endocrine pancreas, due to perturbed islet neogenesis