Actrapid penfill

Blood was collected from the tail and glucose levels were determined using Glucomen aero 2K glucometer (A. Menarini Diagnostics, Florence, Italy). For the glucose tolerance test (GTT), mice were fasted for 6 h before injection of a glucose bolus (2 g/kg) intraperitoneally (IP), and blood glucose levels were measured at 0, 5, 15, 30, 90, and 120 min after injection. For the insulin tolerance test (ITT), mice were fasted for 6 h and then 1 U/kg of human insulin (Actrapid Penfill, Novo Nordisk A/S, Bagsværd, Denmark) was IP-injected. Glucose levels were measured at 15, 30, 90, and 120 min after injection.

After 9 weeks, an oral glucose tolerance test (OGTT) was performed on 4h-fasted mice by giving an oral gavage of glucose (1 g glucose/kg BW, G8769, Sigma-Aldrich, St. Louis, MO). After 10 weeks, an intraperitoneal insulin tolerance test (IITT) was performed on 4h-fasted mice by administering intraperitoneal injections of insulin (0.75 U insulin/kg BW, Actrapid Penfill, Novo Nordisk, Bagsvaerd, Denmark). Blood glucose was assayed in blood samples taken from the tip of the tail before glucose or insulin were administered (at 0 min), and again at 15, 30, 60, and 130 minutes after administration and analyzed using an ACCU-CHEK Aviva and test strips (Roche, Mannheim, Germany). After 11 weeks of treatment, blood was collected from 4h-fasted mice by cardiac puncture during anesthesia with Isoflurane, after which the anesthetized mice were sacrificed by cervical dislocation. Collected organs were weighted and flash-frozen in liquid nitrogen. The Stockholm South Ethical Committee approved this research (Approval number S96-11).

After two weeks of daily stress, mice received intraperitoneal glucose tolerance test (GTT) and insulin tolerance test (ITT) using standard methods^{4 (link)}. Briefly, for GTT, mice were fasted overnight and then challenged with D-glucose at 2 g/kg body weight (Sigma-Aldrich, St. Louis, MO), followed by serial measurements of blood glucose up to 120 min using a blood glucose level monitor (Glutest Ace, Sanwa Kagaku Kenkyusho Co., Nagoya, Japan). For ITT, mice were fasted for 16 hours before testing. Insulin (0.75 U/kg, Actrapid Penfill, NovoNordisk, Copenhagen, Denmark) was injected intraperitoneally, and blood glucose was measured.

At 16 weeks of age, the mice fed with HF/HS were subjected to intraperitoneal glucose tolerance test (GTT) and insulin tolerance test (ITT) using standard protocols, as described in detail previously^{16 (link)}. Briefly, for GTT, the mouse was fasted overnight and then challenged with 2 g/kg d-glucose (Sigma-Aldrich), followed by serial measurements of blood glucose up to 120 min using a blood glucose level monitor (Glutest Ace, Sanwa Kagaku Kenkyusho Co, Nagoya, Japan). For ITT, the mouse was fasted for 16 h before testing. Insulin (0.75 U/kg, Actrapid Penfill, NovoNordisk) was injected intraperitoneally, and blood glucose level was measured serially.

The hypoglycemic clamp was induced after the 30 min of baseline recordings, utilizing a bolus of 15 IU human regular insulin (Actrapid^®, Penfill^®, 100 IU/mL, Novo Nordisk Pharma, Mainz, Germany) via the ear vain catheter, and was maintained between 0.5 and 4 mmol/L over 75 min by additional boluses of insulin. In order to facilitate a continually diminishing blood glucose level, additional boluses of regular insulin were administered if necessary, to mirror the clinical case of an insulin overdose. The individual total insulin dosages varied from 20 to 55 IU of insulin per experiment. A commercially available blood glucose meter (Contour^®, Bayer AG, Leverkusen, Germany) was used to monitor the arterial blood glucose level at baseline and every 7.5 min during the hypoglycemic clamp. Arterial blood samples for ACTH and cortisol measurement were obtained at 30 min of baseline and then every 15 min. After the end of the experiments, the animals were euthanized by intravenous administration of 60 mg/kg pentobarbital (Narcoren^®, Merial, Hallbergmoos, Germany).

Glucose tolerance tests were performed by oral administration of glucose (1 g D-glucose/kg body weight) after overnight fasting. Insulin tolerance tests were performed by intraperitoneal (ip) injection of human insulin (0.75 unit insulin/kg body wt, Actrapid Penfill, NovoNordisk) 5 h after food removal. Pyruvate tolerance tests were performed by ip injection of sodium salt pyruvate (2 g/kg body weight) after an overnight fast. Blood was collected from the tail vein in a heparinized capillary tube and glucose concentration was determined using the One-Touch AccuChek Glucometer (Roche).