Refinement of porcine models in diabetes and transplantation research

Animal models are widely used in biomedical research aiming to prevent and improve treatments of diseases. The 3Rs (replace, reduce, refine) are considered when working with laboratory animals. Socialisation and training of pigs in research are important to avoid stress responses that could potentially affect research data. Domestic pigs were subjected to a structured training programme before inclusion in renal transplantation studies. The training programme enabled blood and urine sampling, and ultrasound examinations in conscious pigs without restraint. The porcine diabetes model was further characterised with regards to hormonal responses to oral glucose, metabolic changes due to insulin deficiency, distribution of glucagon-like peptide-1 receptors (GLP-1R) in pancreas and gastrointestinal tract, and GLP-1R occupancy in vivo during oral glucose tolerance test (OGTT). Both similarities and differences between pigs and humans were identified, both of which are important to keep in mind when designing animal studies. Furthermore, a refined model for OGTT in pigs, which reduces experimental variation and facilitates comparisons between experiments, was established. An insulin treatment protocol for streptozotocin diabetic pigs was developed to enable long-term studies and minimise risk of serious complications. Well characterised animal models and calm animals are important to acquire relevant and reliable research data, and to minimise the number of animals needed. Using refined techniques to minimise stressful situations is also important from an animal welfare perspective. Results presented in this thesis contribute to the 3Rs

The pig as an animal model for type 1 Diabetes Mellitus – with focus on carbohydrate and fat metabolism

Diabetes mellitus is an endocrine disorder affecting a range of different mammals, including dogs, cats, horses and humans. Worldwide, 347 million people have diabetes, and the disease is an increasing burden on the world’s poorest countries. The disease is characterised by chronic hyperglycaemia and afflicted individuals experience increased thirst, polyuria and weight loss, sometimes despite increased appetite. Diabetes mellitus is either insulin dependent or non-insulin dependent. In the present essay, focus is on insulin dependent, type 1 diabetes mellitus (T1DM); a condition where the insulin producing β-cells of the pancreas are destroyed and the insulin producing capacity diminished. Before insulin was discovered and became available as treatment, diabetes was a fatal disease. If T1DM is left untreated, the patient develops diabetic ketoacidosis, a potentially life-threatening condition. During treatment with insulin, there is a risk of insulin-induced hypoglycaemia, also a life-threatening complication. Animal models for diabetes mellitus are important to enable research to gain more information about the disease and to develop new and improved treatments. Rodent models are widely used for diabetes research. However, large animal models are very important tools for preclinical studies. The pig is a suitable large animal for diabetes studies. The physiology and metabolism of the pig resembles that of humans. Diabetes mellitus is a disease of the pancreas, and the size, shape and position of the porcine pancreas, resembles that of the human pancreas. Blood glucose concentrations range within the same levels as in humans and porcine insulin differ by only one amino acid compared to human insulin. Diabetes can occur spontaneously in mice and rats, but have to be experimentally induced in pigs. A type 1 diabetes mellitus like state can be induced with pancreatectomy or by injection of chemicals toxic to β-cells such as streptozotocin (STZ) or alloxan. In the present experiment it is showed that T1DM can be safely induced in domestic pigs at an i.v. STZ dose of 150 mg/kg b.w. The pigs develop clinical signs of diabetes mellitus such as polyuria/polydipsia and reduced weight gain compared to healthy individuals. Already two days post STZ treatment the pigs became hyperglycaemic and a significant (p<0.05) rise in serum non-esterified fatty acids (NEFA) concentrations and serum triglyceride concentrations was seen. Hyperglycaemia, hypertriglyceridaemia and elevated serum NEFA concentrations are also seen in diabetic humans. One week after initiation of insulin treatment, normoglycaemia is restored. One or three weeks after initiation of insulin treatment, serum concentrations of NEFA and triglycerides, respectively, are lowered to levels no different from that seen in the pigs before the induction of diabetes. This lowering of glucose, NEFA and triglyceride levels are also seen in insulin treated human patients with T1DM. From the present experiment it is concluded that a daily dosage of 1 IU/kg b.w. of short-acting insulin, divided by two times and given in direct connection to feeding is a satisfying treatment regimen. Both non-insulin treated and insulin treated STZ-diabetic pigs are brisk and vivid, and have good appetite.Diabetes mellitus är en endokrin sjukdom som drabbar flera olika däggdjur såsom hund, katt, häst och människa. 347 miljoner människor runt om i världen har diabetes och sjukdomen börjar bli en allt större börda i några av världens fattigaste länder. Sjukdomen karaktäriseras av kronisk hyperglykemi och drabbade individer får ökad törst, ökad urinmängd och rasar i vikt, ibland trots ökad aptit. Diabetes mellitus är antingen insulinberoende eller icke-insulinberoende. I denna uppsats ligger fokus på insulinberoende, typ-1 diabetes (T1DM), en sjukdom där de insulinproducerande β-cellerna i bukspottkörteln förstörs och den insulinproducerande förmågan minskar. Innan insulin upptäcktes och blev tillgängligt som behandling dog diabetiker i ung ålder. Om T1DM lämnas obehandlat utvecklar patienten diabetisk ketoacidos, ett livshotande tillstånd. Under insulinbehandling finns det alltid en risk för insulin-inducerad hypoglykemi, även detta en livshotande komplikation. Djurmodeller är viktiga redskap i diabetesforskningen för att få mer information om sjukdomen och för att utveckla nya och förbättra behandlingsalternativ. Gnagare används mycket inom diabetesforskning, men stordjursmodeller är mycket viktiga i pre-kliniska studier. Grisen är en lämplig stordjursmodell för diabetesforskning eftersom grisens fysiologi och metabolism liknar människans. Diabetes mellitus är en sjukdom i bukspottkörteln och storleken, utseendet och läget på grisens bukspottkörtel liknar människans bukspottkörtel. Grisens blodglukoskoncentration varierar inom samma intervall som hos människa och porcint insulin skiljer sig bara med en aminosyra från humant insulin. Möss och råttor kan spontant få diabetes, men när man använder grisar måste diabetes induceras experimentellt. Ett tillstånd som liknar T1DM kan induceras genom pancreasektomi eller genom injektion av substanser som är toxiska för β-cellerna, såsom streptozotocin (STZ) eller alloxan. I den här studien visas att diabetes kan induceras säkert i konventionella svenska grisar med en intravenös dos på 150 mg/kg kroppsvikt av STZ. Grisen utvecklar symtom på diabetes mellitus såsom polyuri/polydipsi och minskad tillväxt jämfört med friska grisar. Redan två dagar efter STZ-behandlingen blir grisarna hyperglykemiska och får en signifikant (p<0.05) ökning av serumkoncentration av NEFA och triglycerider. Hyperglykemi, hypertriglyceridemi och ökade nivåer av NEFA i serum ses också hos människor med diabetes mellitus. En vecka efter att insulinbehandlingen startade blev grisarna återigen normoglykemiska. En respektive tre veckor efter insulinbehandlingens påbörjan sjönk serumnivåerna av fria fettsyror (NEFA) respektive triglycerider till nivåer som ej skiljde sig från nivåerna som sågs hos grisarna innan diabetesinduktionen. Dessa sänkningar av blodglukos, NEFA-koncentrationer och triglyceridkoncentrationer ses även hos insulinbehandlade patienter med T1DM. Från den här studien kan konkluderas att en daglig dos på 1 IE/kg kroppsvikt kortverkande insulin, uppdelat på två gånger, givet i direkt samband med utfodring är en tillfredställande behandlingsregim. Både insulinbehandlade och icke-insulinbehandlade STZ-diabetiska grisar är livliga, pigga och har god aptit

A battery-less implantable glucose sensor based on electrical impedance spectroscopy

The ability to perform accurate continuous glucose monitoring without blood sampling has revolutionised the management of diabetes. Newer methods that can allow measurements during longer periods are necessary to substantially improve patients' quality of life. This paper presents an alternative method for glucose monitoring which is based on electrical impedance spectroscopy. A battery-less implantable bioimpedance spectroscope was designed, built, and used in an in vivo study on pigs. After a recovery period of 14 days post surgery, a total of 236 subcutaneous bioimpedance measurements obtained from intravenous glucose tolerance tests, with glucose concentration ranges between 77.4 and 523.8 mg/dL, were analyzed. The results show that glucose concentrations estimated by subcutaneous bioimpedance measurements correlate very well to the blood glucose reference values. The pigs were clinically healthy throughout the study, and the postmortem examinations revealed no signs of adverse effects related to the sensor. The implantation of the sensor requires minor surgery. The implant, being externally powered, could in principle last indefinitely. These encouraging results demonstrate the potential of the bioimpedance method to be used in future continuous glucose monitoring systems

Marine Strategy Framework Directive - Task Group 11 Report Underwater Noise and Other Forms of Energy

The Marine Strategy Framework Directive (2008/56/EC) (MSFD) requires that the European Commis-sion (by 15 July 2010) should lay down criteria and methodological standards to allow consistency in approach in evaluating the extent to which Good Environmental Status (GES) is being achieved. ICES and JRC were contracted to provide scientific support for the Commission in meeting this obligation. A total of 10 reports have been prepared relating to the descriptors of GES listed in Annex I of the Directive. Eight reports have been prepared by groups of independent experts coordinated by JRC and ICES in response to this contract. In addition, reports for two descriptors (Contaminants in fish and other seafood and Marine Litter) were written by expert groups coordinated by DG SANCO and IFREMER respectively. A Task Group was established for each of the qualitative Descriptors. Each Task Group consisted of selected experts providing experience related to the four marine regions (the Baltic Sea, the North-east Atlantic, the Mediterranean Sea and the Black Sea) and an appropriate scope of relevant scien-tific expertise. Observers from the Regional Seas Conventions were also invited to each Task Group to help ensure the inclusion of relevant work by those Conventions. This is the report of Task Group 11 Underwater noise and other forms of energy.JRC.DDG.H.5-Rural, water and ecosystem resource

Exploring the GLP-1-GLP-1R axis in porcine pancreas and gastrointestinal tract in vivo by ex vivo autoradiography

Introduction Glucagon-like peptide-1 (GLP-1) increases insulin secretion from pancreatic beta-cells and GLP-1 receptor (GLP-1R) agonists are widely used as treatment for type 2 diabetes mellitus. Studying occupancy of the GLP-1R in various tissues is challenging due to lack of quantitative, repeatable assessments of GLP-1R density. The present study aimed to describe the quantitative distribution of GLP-1Rs and occupancy by endogenous GLP-1 during oral glucose tolerance test (OGTT) in pigs, a species that is used in biomedical research to model humans.Research design and methods GLP-1R distribution and occupancy were measured in pancreas and gastrointestinal tract by ex vivo autoradiography using the GLP-1R-specific radioligand Lu-177-exendin-4 in two groups of pigs, control or bottle-fed an oral glucose load. Positron emission tomography (PET) data from pigs injected with Ga-68-exendin-4 in a previous study were used to retrieve data on biodistribution of GLP-1R in the gastrointestinal tract.Results High homogenous uptake of Lu-177-exendin-4 was found in pancreas, and even higher uptake in areas of duodenum. Low uptake of Lu-177-exendin-4 was found in stomach, jejunum, ileum and colon. During OGTT, there was no increase in plasma GLP-1 concentrations and occupancy of GLP-1Rs was low. The ex vivo autoradiography results were highly consistent with to the biodistribution of Ga-68-exendin-4 in pigs scanned by PET.Conclusion We identified areas with similarities as well as important differences regarding GLP-1R distribution and occupancy in pigs compared with humans. First, there was strong ligand binding in the exocrine pancreas in islets. Second, GLP-1 secretion during OGTT is minimal and GLP-1 might not be an important incretin in pigs under physiological conditions. These findings offer new insights on the relevance of porcine diabetes models

Adolescents with obesity treated with exenatide maintain endogenous GLP-1, reduce DPP-4, and improve glycemic control

BackgroundGLP-1 receptor agonists (GLP-1RA) are increasingly used to treat adolescent obesity. However, the effect on endogenous GLP-1 secretory patterns following treatment in adolescents is unknown. The GLP-1RA exenatide was shown to significantly lower BMI and 2-hour glucose in adolescents with obesity, in the placebo-controlled, randomized controlled trial Combat-JUDO. The aim of this study was to evaluate effects of weekly injections of 2 mg exenatide extended release on secretory patterns of endogenous hormones during OGTT.Subjects and MeasurementsThis study was a pre-planned sub-study of the Combat-JUDO trial, set at the Pediatric clinic at Uppsala University Hospital, Sweden and Paracelsus Medical University, Austria. 44 adolescents with obesity were included and randomized 1:1 to treatment:placebo. 19 patients in the treatment group and 18 in the placebo group completed the trial. Before and after treatment, GLP-1, glucose, insulin, glucagon and glicentin levels were measured during OGTT; DPP-4 and proinsulin were measured at fasting. A per-protocol approach was used in the analyses.ResultsExenatide treatment did not affect GLP-1 levels during OGTT. Treatment significantly lowered DPP-4, proinsulin and the proinsulin-to-insulin ratio at fasting, increased glicentin levels but did not affect insulin, C-peptide or glucagon levels during OGTT.ConclusionWeekly s.c. injections with 2 mg of exenatide maintains endogenous total GLP-1 levels and lowers circulating DPP-4 levels. This adds an argument in favor of using exenatide in the treatment of pediatric obesity.Clinical trial registrationclinicaltrials.gov, identifier NCT0279440

Dissecting the physiology and pathophysiology of glucagon-like peptide-1

Copyright © 2018 Paternoster and Falasca. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. An aging world population exposed to a sedentary life style is currently plagued by chronic metabolic diseases, such as type-2 diabetes, that are spreading worldwide at an unprecedented rate. One of the most promising pharmacological approaches for the management of type 2 diabetes takes advantage of the peptide hormone glucagon-like peptide-1 (GLP-1) under the form of protease resistant mimetics, and DPP-IV inhibitors. Despite the improved quality of life, long-term treatments with these new classes of drugs are riddled with serious and life-threatening side-effects, with no overall cure of the disease. New evidence is shedding more light over the complex physiology of GLP-1 in health and metabolic diseases. Herein, we discuss the most recent advancements in the biology of gut receptors known to induce the secretion of GLP-1, to bridge the multiple gaps into our understanding of its physiology and pathology