Humulin r

The rats fasted for a 14 h period in advance (18:00 to 8:00) of the experiment. After intramuscular anesthesia was administered, cannula of the right jugular vein and left common carotid artery was carried out. Intravenous infusion channels and three-limb tubes were established, of which one end was connected to the insulin pump, and the other end was connected to the glucose injection pump. The basal blood glucose was required to be measured continuously 3 times, with intervals of 5 min each time after the blood glucose was stable. The infusion of human insulin (Humulin R, Novo Nordisk) was conducted at a constant rate of 4 mU · kg⁻¹ · min⁻¹, with the blood glucose measurement time of 5 min for each interval. The rate of the glucose infusion was adjusted according to the measured blood glucose value, making sure that there was stability in the blood glucose value (basal blood glucose value ± 0.5) mmol/L. When 3 consecutive glucose values were all in the above range, the evaluation index of the insulin sensitivity of the rats was the average value of the GIR in 60 to 120 min.

After 16 weeks of feeding, the clamp was done. The rats were anesthetized with isoflurane (Sinopharm Chemical Reagent Beijing Co., Ltd, China) after fasting overnight. The left common carotid artery and right jugular vein were catheterized as previously described [39 ]. The clamps were then performed 4–5 d later after complete recovery of the rats from the operation. Rats (n =4 per group) fasted for 12 h, were infused with human insulin (Humulin R, Novo Nordisk) into the venous circulation, at a rate of 4 mU · kg-1 min-1 for 2 h. Throughout the infusion, the carotid artery was assessed every 10 min using a blood glucose meter(One Touch Uitra, Lifescan). Titration of glucose continues until stable glucose readings are achieved. Stable glucose levels over a time course of a minimum of 30 min. Glucose levels and glucose infusion rates during this stable period are recorded and reported. Results provide an index of whole body insulin sensitivity.

Glucose tolerance test (GTT) and insulin tolerance test (ITT) were conducted at the 6th week. GTT procedure was reported previously^{35 (link)}. ITT was carried out as follows: Mice were fasted for 4 h (9:00 A.M. to 1:00 P.M.) and insulin (1 U/kg Humulin R; Novo Nordisk) was administered intraperitoneally. The blood glucose levels were measured immediately before and after 15, 30, 45, and 60 min of insulin injection with an Accu-Chek glucose monitor (Roche Diagnostics, Indianapolis, IN, USA).

Initially, a time course study was undertaken using 16 rats to determine the optimal time for analysis of mechanically-induced signaling events after ulna loading. Eight time points of 0, 15, 30 minutes, 1, 2, 6, 12, and 24 hours after loading were selected based on previous mechanical signaling events in other tissues and existing in vitro literature (n = 2 rats/time point). At each time point, the right ulna of one rat underwent mechanical loading and the second rat was sham-loaded. Rats were randomly chosen from the colony for group designation. An additional three rats were treated with insulin (5 IU/kg i.p., Humulin R, Novo Nordisk, Princeton, NJ) 15 minutes before euthanasia to serve as positive controls for the phosphorylation kinases in the PI3K/Akt/mTOR and MAPK signaling pathways [40 (link)].
Based on data from the time course study, time points of 15 minutes, and 1, 6, 12 and 24 hours after ulna loading were selected as the most informative regarding analysis of mechanically-induced signaling events after ulna loading. An additional 48 rats were then used for these time points. Load and sham group sizes were n = 3, respectively, at 15 minutes and 1 hour, and n = 6 at 6, 12, and 24 hours.

The activity, diet, and body posture of rats were recorded. Weekly body weight monitoring, FBG (fasting blood glucose), and RBG (random blood glucose) were measured at 7am to 9 am.
OGTT was measured at the sixth week of administration, and the area under the curve (AUC) was calculated. The rats were fasted for 12 h, and 50% glucose solution was used for intragastric administration according to 2 g/kg criteria. Blood glucose value was measured with the intragastric administration before (0 min) and 30, 60, and 120 min after. The formula for AUG calculation for blood glucose (BG) levels observed during the OGTT is as follows: AUC = 0.5 × (BG 0 min + BG 30 min)/2 + 0.5×(BG 30 min + BG 60 min)/2 + 1 × (BG 60 min + BG 120 min)/2.
Rats were given the ITT test on the last day of 6 weeks of administration, and 2 U/kg insulin (Humulin R, Novo Nordisk, Denmark) was subcutaneously injected in rats, and hypoglycemic effect was observed at 0 min, 30 min, 60 min, and 120 min. Also AUC was calculated according to the above formula.