Alphatrak

Blood glucose was measured hourly for the first 24 h using two portable blood glucose meters previously validated for use in dogs (Optium Xceed, Abbott, UK; AlphaTRAK, Zoetis, USA) (31 (link)–34 (link)), and then every 2–3 h during the intensive insulin protocols. Blood BHB was monitored every 4 h using a portable ketometer, previously validated for dogs (Optium Xceed, Abbott, UK) (35 (link)), until the BHB was ≤1.0 mmol/L. The capillary blood samples for the measurement of BG and BHB concentrations were obtained using needles or lancing devices on the inner aspect of the pinna. A venous blood gas analysis, including an electrolyte panel, was carried out, within 5 min of collection, using a blood gas analyzer (ABL 800 Flex, Radiometer Medical ApS, Brønshøj, DK) every 8 h during the first 24 h, and then every 12 h until the acidemia was resolved. Serum phosphate was measured using an automated chemistry analyzer (AU400, Beckman-Coulter/Olympus, O'Callaghan's Mills, Ireland) every 8 h during the first 24 h, and then every 12 h until the DKA was resolved. The venous blood samples were collected by jugular, cephalic or femoral phlebotomy.
Insulin-induced hypoglycemia was defined as a BG concentration <80 mg/dL; hypokalemia and hypophosphatemia were defined as serum potassium ≤3.6 mEq/L and serum phosphate ≤2.65 mg/dL, respectively.

GTTs were performed in 10-week-old male C57/Bl6 mice (mean weight: IPGTT 27.7 ± 0.2 g, OGTT 25.5 ± 0.3 g) after a 6 h fast, using n = 8 per group determined by power analysis where the effect size was 15%, and 80% power and significance <0.05. Depending on the group (detailed in Results), animals were dosed subcutaneously with antibody or saline 24 h prior to the GTT.
For the IPGTT, mice were dosed with 0.1 mg/kg liraglutide (Victoza; Novo Nordisk, Gatwick, UK) or vehicle subcutaneously 2 h before intraperitoneal glucose administration (2 g/kg). Blood glucose levels were determined from tail-prick blood samples using a hand-held glucometer (AlphaTrak; Zoetis, London, UK) at −120, 0, 15, 30, 45, 60, 90 and 120 min relative to the glucose challenge. For the OGTT, mice were orally gavaged with glucose (2 g/kg), and blood glucose levels were determined at 0, 15, 30, 45, 60, 90 and 120 min relative to the glucose challenge. At the end of the experiment, animals were euthanised using cervical dislocation. The AUC between 0 and 120 min was calculated, and statistical significance was assessed by one-way ANOVA with post hoc Bonferroni test.

Following blood sampling EDTA tubes were placed immediately in crushed ice and serum tubes were allowed to clot at ambient temperature. Two to four hours following blood collection, tubes were centrifuged at 3,000 g for 10 min in a laboratory. Serum insulin concentration and, in animals aged ≥10 years, plasma ACTH concentration were measured in duplicate using chemiluminescent assays (Immulite 2000/Immulite 2000XPi, Siemens, Healthcare) previously validated in horses.¹⁷ (link), ¹⁸ (link) Samples with an insulin concentration greater than the upper reportable limit (300µIU/mL) were diluted with charcoal‐stripped serum.¹⁷ (link) The model of the chemiluminescent analyser was updated from the Immulite 2000 to the Immulite 2000XPi after 234 animals. To adjust for the new analyser paired measurement of insulin concentration in 39 samples was performed. Regression analysis (Supplementary Item 2) was used to transform results from the Immulite 2000 to the newer Immulite 2000XPi, which are presented here. Blood glucose concentration was measured using a validated point of care glucometer (AlphaTRAK, Zoetis).¹⁹ (link)

ITT and GTT assays were as described in^{21 (link),22 (link)}. Prior to GTT, mice were fasted for 5 h. Basal glucose levels were determined by glucometer readings (AlphaTRAK, Zoetis) from tail punctures. Mice were intraperitoneal injected with a 2 mg/g d-glucose (20%, Sigma-Aldrich) bolus. Blood glucose was monitored at 15, 30, 60 and 90 min post-injection. Mice had ad libitum excess to water throughout. ITT was performed by intraperitoneal injection of 0.75 mU/g insulin (Hypurin Bovine Neutral, Wockhardt) per gram of body weight of fasted (5 h) mice.

Cohorts of 5- to 6-month-old female mice, wild-type (n = 5) and Bglap/2^dko/dko (n = 5), were subjected to four random glucose measurements taken 6 h into their light cycle by tail nick using a glucose meter (AlphaTRAK; Zoetis) on four consecutive days. These same cohorts had glucose measurements obtained by tail nick after an overnight fast during which the animals had access to water on two occasions one week apart.
Additional cohorts of mice were fasted overnight, but with access to water, prior to euthanasia by CO₂ inhalation. Glucose levels were measured immediately after euthanasia. Animals were euthanized by CO₂ inhalation and glucose levels were immediately measured from blood collected by tail nick using a glucose meter.
Glucose data were analyzed using a linear mixed-effects model via the R package lme4 [37 (link)] to account for repeated sampling. Normality of the residuals was verified visually using a qq-plot.