Actrapid

Sixteen-week-old male C57Bl/6 mice were separated into one of four groups (six per group) to receive morning injections of saline (PBS), insulin (daily; 0.75 U/kg/BW; Actrapid; Novo Nordisk, Plainsboro, NJ), myriocin (thrice weekly; 0.3 mg/kg; Sigma) or both for 28 days with free access to water and chow (Harlan 8604) throughout the length of the study. After the 28-d treatment, mice underwent intraperitoneal glucose (G7021; Sigma-Aldrich, St. Louis, MO) and insulin (Actrapid; Novo Nordisk, Plainsboro, NJ) tolerance tests. For both tests, mice were fasted for 6 h and received an injection of either glucose (1 g/kg body wt) or insulin (0.75 U/kg body wt). These are doses that are above the typical rate of insulin treatment in type 2 diabetics (0.5 U/kg) [28 (link)]. Plasma glucose (Bayer Contour glucose meter), insulin (ELISA; Crystal Chem Inc.), and adiponectin (Crystal Chem Inc.) levels were determined. Studies were conducted in accordance with the principles and procedures outlined in the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the IACUC (Institutional Animal Care and Use Committee) at Brigham Young University.

Myoblast survival was measured by tryphan blue staining (Sigma-Aldrich) immediately after shockwave treatment. In order to examine the proliferation rate after Li-SWT, myoblasts were cultured in 10 mM BrdU in UG media (cumulative BrdU incorporation), and coverslips were harvested at days 1, 2, 3 and 4 after treatment. The differentiation ability of myoblasts after Li-SWT was tested by culturing them in a differentiation medium (DMEM with 2% FBS, 1% PSA and 25 pM insulin (Actrapid from Novo Nordisk, DK)) and cells were harvested on days 2, 4, and 6 after Li-SWT.

Peripheral insulin sensitivity was determined in a subgroup of overweight/obese, prediabetic individuals via hyperinsulinaemic-euglycemic clamps as previously described^{57 (link),58 (link)}. In short, a cannula was inserted into an antecubital vein for infusion of glucose and insulin. To measure blood glucose, a second cannula was inserted into a superficial dorsal hand vein, which was arterialized by placing the hand into a hotbox (~50 °C). A priming dose of insulin infusion (Actrapid, Novo Nordisk, Gentofte, Denmark) was administered during the first ten min (t0–t10 min) and insulin infusion was thereafter continued at 40 mU/m²/min for 2 h (t10–t120 min). By variable infusion of a 20% glucose solution, plasma concentrations were maintained at 5.0 mmol/L. Peripheral insulin sensitivity (M-value, mg*(kg*min)⁻¹ was calculated from the mean glucose infusion rate during the steady-state of the clamp (last 30 min, stable blood glucose concentration at 5.0 mmol/L)^{62 (link)}. A high M-value represents high insulin sensitivity (i.e., more glucose needs to be infused to maintain euglycemia during insulin infusion).

To test acute effects of JM-00266 on glucose metabolism and their specificity to CB1R, C57BL/6 wild type mice and CB1R^−/− mice fasted for 6 h were subjected to an oral glucose tolerance test (OGTT, 2 g·kg⁻¹ glucose) and to an insulin tolerance test (ITT) using 0.5 UI·kg⁻¹ insulin (Actrapid, Novo Nordisk, La Défense, France) 10 min after i.p. injection of JM-00266 (10 mg·kg⁻¹) or vehicle. For both tests, glycemia was monitored during 120 min post-injection with a My Life Pura TM glucose meter (Ypsomed, Paris, France) directly on a blood drop from the tail. Corresponding AUC_{0–2 h} were calculated.

Mice received a subcutaneous injection of insulin glargine (lantus, 1 unit/kg body weight) or vehicle solution (0.9% sterile NaCl) 2 h before the night-active phase (5 p.m.) for four consecutive days in a crossover design 5 days apart. Glucose levels were sampled from mouse tail bleeds at 9 a.m. and 5 p.m. using a Glucometer Elite (Bayer Health Care, Leverkusen, Germany). To exclude inter-individual variation in cortical ECoG activity, the data for the ECoG power density are expressed as percentage change from vehicle application. An intracerebroventricular (i.c.v.) injection with human insulin (actrapid, Novo Nordisk, Denmark) in a concentration of 3.75 mU/ 5 μL (which correspond to 5.41 nmol/mL) was performed according to published literature [37 (link)–39 (link)] and as previously described [4 (link)].