Glucocard g

Glycaemia and the serum levels of metabolic hormones and lipids were assessed after 14 days of treatment and at sacrifice.
Glycaemia was measured using the glucometer Glucocard G (Arkray Factory, Inc.; Shiga, Japan). Insulin, leptin, and adiponectin serum levels were determined by ELISA kits according to manufacturer’s instructions (Merck Millipore, Dramstadt, Germany). The Homeostatic Model Assessment of Insulin Resistance (HOMA-IR) index was calculated through the following formula: fasting glucose (mg/dL) × (fasting insulin (ng/mL)/405). Serum levels of total lipids, triglycerides, total cholesterol, and low-density lipoprotein (LDL) and high-density lipoprotein (HDL) cholesterol were determined using colorimetric assays from Spin React (Sant Esteve de Bas, Girona, Spain).

The OGTT and IPITT were performed according to a previously reported method [47 (link)]. Briefly, mice were fasted for 16 h (overnight fasting) for the OGTT and 4 h (morning fasting) for the IPITT at weekly intervals. Using the GLUCOCARD G+ (Arkray, Kyoto, Japan), blood glucose levels were measured 0, 15, 30, and 60 min after oral glucose (1 g/kg body weight) administration and intraperitoneal insulin (0.75 units/kg) injection. A 35-µL blood sample was collected from the tail vein using a heparinized plastic hematocrit tube (Drummond Scientific Company, Broomall, PA, USA), and plasma was separated by centrifugation at 2500× g for 30 min. After centrifugation, the plasma was stored at −80 °C for future insulin measurement. The Rebis Insulin-mouse U ELISA kit (Shibayagi, Gunma, Japan) was used to measure insulin levels. The AUC for blood glucose was calculated using the linear trapezoidal method for both the OGTT and IPITT.

Half of the old rats (Old) and young rats were administered with water for 21 days while the other half of old rats were (Old + Oil Mixture + OLE) administered with OLE in drinking water and treated orally with 2.5 mL/Kg of the Oil Mixture (75% of EVOO and 25% of AO) once daily. In order to avoid degradation, OLE was renewed every 3 days and the dosage was adjusted to the liquid intake and the animal’s body weights.
Over the three week treatment, body weight was measured daily and food intake and water intake were measured weekly. After the treatment period and an overnight fasting, the animals were killed by decapitation after an overdose of sodium pentobarbital (100 mg/Kg). Trunk blood was collected and the serum was obtained after centrifugation at 3000 rpm for 20 min. Glycemia was measured before sacrifice by venous tail puncture using the glucometer Glucocard G™ (Arkray Factory, Inc.; Shiga, Japan). Immediately after, the subcutaneous (lumbar), visceral (epididymal), perivascular (aortic) and brown (interscapular) adipose tissue depots, as well as the heart, liver, spleen, kidneys, adrenal glands and gastrocnemius and soleus muscles were dissected, weighed and stored at −80 °C for further analysis.