Intraperitoneal and subcutaneous glucagon delivery in anaesthetized pigs: effects on circulating glucagon and glucose levels

Intraperitoneal and subcutaneous glucagon delivery in anaesthetized pigs: effects on circulating glucagon and glucose levels

Animals and animal handlingBetween February and June 2018, eleven female, juvenile, non-diabetic farm pigs (Sus scrofa), weighing 34–50 kg, were brought to the animal research facility approximately one week before experiments and acclimated to the staff and new environment. They were housed together in a common pen, in groups of two or three whenever possible, provided wood chips as nesting material and toys to keep them occupied. The lighting condition was standardized with a 16 h light period followed by an 8 h dark period. They were fed standard commercial growth feed twice a day and provided water ad libitum. Food was removed nine-ten hours prior to the experiments.AnaesthesiaThe pigs were premedicated with an IM injection of 4 mg diazepam (Stesolid®, Actavis Group, Hafnarfjordur, Iceland), 160 mg azaperone (Stresnil®, Eli Lilly Regional Operations GmbH, Austria) and 750 mg ketamine (Ketalar®, Pfizer AS, Norway), while in the pen. An ear vein was cannulated, and anaesthesia was induced with an IV injection of 1 mg atropine (Takeda AS, Asker, Norway), 150–250 µg fentanyl (Actavis Group, Hafnarfjordur, Iceland), 75–125 mg thiopental (VUAB Pharma AS, Roztoky, Czech Republic) and 150–250 mg ketamine (Ketalar®, Pfizer AS, Norway).The pigs were intubated in the lateral position and mechanically ventilated and monitored on an anaesthesia machine (Aisys, GE Healthcare Technologies, Oslo). Anaesthesia was maintained by IV infusion of midazolam (0.5 mg/kg/h) (Accord Healthcare Limited, Middlesex, UK) and fentanyl (7.5 µg/kg/h) (Actavis Group, Hafnarfjordur, Iceland) and by inhalation of isoflurane (0–2%) (Baxter AS, Oslo, Norway). The room temperature was around 20 °C. The body temperature of the pigs was continuously monitored, and a heating blanket applied when necessary.The pigs received IV infusions of antibiotics (Cefalotin, Villerton Invest SA, Luxembourg), 2 g immediately after the pigs were anaesthetized and 1 g after 4 h. Heparin (150 IE) (LEO Pharma A/S, Ballerup, Denmark) was injected in the peritoneal space at the same time points.Fluid balance was achieved by continuous IV infusion of Ringer’s acetate, approximately 1,000 ml during the length of the experiment, with individual adjustments. The described anaesthesia protocol have been used in previous pig experiments37.Surgical procedureAn intra-arterial line was placed in the left carotid artery for blood sampling and monitoring of physiological parameters and an IV line was placed in the left internal jugular vein for fluid infusions. Both catheters were inserted through the same cut-down.The catheter from an Animas Vibe insulin pump (serial number 80-26400-16, Animas Corp., West Chester, PA, USA) was inserted 10–15 cm into the upper left part of the abdomen through a 2–3 cm long craniocaudal incision in the abdominal wall, 2–3 cm caudally to the umbilicus. The bladder was exposed through a small, low laparotomy for the insertion of a bladder catheter. Both cuts were made with a thermocauter to minimize bleeding.At the end of the experiments, and under full anaesthesia, the pigs were euthanised with an IV overdose of pentobarbital (minimum 100 mg/kg) (pentobarbital NAF, Apotek, Lørenskog, Norway).Suppression of endogenous glucagon secretionTo inhibit endogenous insulin and glucagon secretion, the pigs were given IV injections of 0.4 mg octreotide (Sandostatin 200 µg/ml, Novartis Europharm Limited, United Kingdom) every hour and SC injections of 0.3 mg pasireotide (Signifor 0.3 mg/ml, Novartis Europharm Limited, United Kingdom) every third hour. To verify glycaemic stability, three blood samples were collected within the last 20 min before the first injection of somatostatin analogues. Suppression efficiency was evaluated by analysing glucagon and insulin levels for 30 min after somatostatin analogue injection (every 10 min). Blood samples were also analysed for porcine insulin at intervals throughout the experiments.The effectiveness of the somatostatin analogues for the suppression of glucagon and insulin secretion is described in the last paragraph of the Results section and in Supplementary Table S2.Glucagon bolusesGlucagon (Novo Nordisk, Denmark) was mixed according to specification and placed in the pump. The volume of one unit by the pump was measured to 10 µl, and 1 unit was equivalent to 10 µg of glucagon. The glucagon in the pump was stored at room temperature for the length of the one-day experiments. Glucagon for the SC dose and the last 1 mg IP dose were mixed just before administration.One pig was used to define the doses of glucagon to be used in the experimental protocol and not included in the analysis. We aimed at boluses which would raise the BGL with one to three mmol/L and chose to investigate three different IP doses of glucagon (0.3 µg/kg, 0.6 µg/kg and 1 mg) and one SC dose (0.6 µg/kg). The 0.3 µg/kg IP, 0.6 µg/kg IP and 0.6 µg/kg SC doses were delivered in a randomized order, with approximately 100 min between each bolus, while the 1 mg IP bolus was given as the last bolus to test maximum glucose effect of IP glucagon delivery. Two pigs, which did not show any post glucagon elevations in BGL, received a 1 mg IV dose at the end of experiments.Glucose analysisArterial blood samples for glucose analysis were collected every 2–15 min throughout the length of experiments, with the highest frequency from 15 before to 80 min after glucagon boluses. Samples were collected in heparinized syringes (LEO Pharma A/S, Ballerup, Denmark) and analysed on a Radiometer ABL 725 blood gas analyser (Radiometer Medical ApS, Brønshøj, Denmark). Most samples were analysed immediately, but some samples were stored on ice for a maximum of 20 min before analysis. We have previously published that such samples can be stored on ice for up to six hours without significant effect on measured glucose values37.Glucagon and porcine insulin analysisThree arterial blood samples within the last 20 min before glucagon boluses were analysed for glucagon and insulin in all pigs. However, arterial blood samples, with the same intervals as glucose samples described above, were only collected from 8 pigs, in total after nine IP boluses and four SC boluses. Arterial blood samples were collected in empty syringes and immediately transferred to EDTA vacutainers (2 ml). The samples were stored in ice water for 10 min before they were centrifuged and the plasma transferred to Eppendorf tubes and stored at − 80 °C until analysis. Glucagon was analysed with Glucagon ELISA (10-1281-01 Mercodia, Uppsala, Sweden) and porcine insulin was analysed with Porcine Insulin ELISA (10-1200-01, Mercodia, Uppsala, Sweden). The assay ranges for the glucagon and porcine insulin ELISA kits were 2–172 pmol/L and 2.3–173 mU/L, respectively.All glucagon samples were run in singles with a coefficient of variability (CV) < 10%. Inter-assay CV were 8%, 8% and 6% for 42.6, 14.7 and 4.98 pmol/L standards, respectively. This glucagon ELISA kit cannot differentiate between endogenous and exogenous glucagon.Statistical analysisThe relationship between BGL and time was analysed for all interventions using a mixed linear model with bolus order and the combination of time and treatments as the fixed effects. The dependent variable was defined as log BGL to achieve normal distribution. To account for multiple measurement series on each pig, pig identification was included as a random effect44. To account for dependence within each series, the error term for each series was specified as a first-order autoregressive process (AR (1)) series accounting for minutes between measurements. The estimated mean changes in BGL, from the mixed linear model, for time points 0–80 min for the 0.6 µg/kg IP, 0.3 µg/kg IP and 0.6 µg/kg SC boluses and time points 0–20 min for the 1 mg IP bolus were compared using the Wald test. The area under the curve (AUC) for delta values from minute 0 to 80 and time until maximum BG change for the different boluses were compared using the Mann–Whitney U test. If the maximum value for BG change was observed for more than one time point, the first occurring time point was selected. The level of significance was set to 0.05. All statistical analyses were carried out in R45.Ethical approvalThe animal experiments were approved by the Norwegian Food Safety Authority (FOTS number 12948) and were in accordance with “The Norwegian Regulation on Animal Experimentation” and “Directive 2010/63/EU on the protection of animals used for scientific purposes”.

Via Source link