Onetouch glucometer

Glucose tolerance was assessed by IPGTT after mice fasted for 12–16 hrs. A bolus of glucose (2 g/kg) was injected intraperitoneally, and blood samples were collected from the tail vein at 0, 15, 30, 60 and 120 min and glucose was measured using a One touch Glucometer (Life‐Scan, Milpitas, CA, USA).

Glucose tolerance test (GTT) and insulin tolerance test (ITT) in all groups were performed largely as described58 (link). Briefly, after overnight and 5-h fasting, GTT and ITT were conducted at ZT2 and ZT 8 respectively. Glucose levels were measured from tail blood before and 15, 30, 60, or 120 min after i.p. injection of either 1 g/kg glucose or 0.75 U/kg insulin (Eli Lilly) by using the ONETOUCH glucometer (LifeScan).
For insulin challenge, mice were given vehicle (PBS) or insulin (5 U/kg) via i.p following overnight fasting (15–18 h). Five minutes after injection, mice were deeply anesthetized by isoflurane inhalation. Isolated liver samples were immediately frozen in liquid nitrogen and stored in −80 °C freezer until later use.

STZ was dissolved in 0.05 M citrate buffer (pH 4.5) and injected intraperitoneally (i.p.) to overnight fasted mice at a single dose of 125 mg/kg body weight. The animals were allowed to drink 5% glucose solution to overcome the drug induced hypoglycemia. After 72 h of STZ administration, blood samples were collected from tail and glucose levels were estimated by glucostrips (One Touch Glucometer, Life Scan, Europe). Mice having fasting blood glucose levels above 200 mg/dl were considered diabetic and subsequently used in the present study.

On the day of electrophysiological recording, blood glucose levels were monitored (One‐touch glucometer, LifeScan Inc, catalog # AW 06398902A) and then animals were anaesthetized under 100 mg/kg thiobutabarbital (inactin) and mounted into a stereotaxic frame. Body temperature was maintained at 37°C throughout surgery and subsequent electrophysiological experimentation via animal placement upon a heating pad. A midline incision was made to expose the underlying skull. A small craniotomy ~1.5 mm in diameter was made over the left or right hemisphere, to allow access to the VMH (centred 2.8 mm posterior and 0.5 mm lateral of Bregma), and a second 1 mm crantiotomy was made 4‐5 mm posterior of Bregma and 2‐4 mm lateral of the midline in the contralateral hemisphere where a cranial screw was placed to serve as ground/reference. A recovery period from surgery of 20 minutes was allowed after the initiation of electrode placement and the beginning of neuronal electrical recordings. Placement of the cannula and recording electrode was verified by electrolytic induction of microlesions at the VMH by passing 150 µA DC for 60 seconds through recording electrodes. Frozen sections were stained with haematoxylin and compared to a rat brain atlas for verification.³⁰ Following the termination of electrophysiological recordings, animals were sacrificed by anaesthesia overdose.

Intraperitoneal glucose tolerance test (IPGTT) was performed to access glucose tolerance after mice fasted for 12 hours. A bolus of glucose (2 g/kg) was injected intraperitoneally. Blood glucose levels were obtained from the tail vein and measured with the OneTouch Glucometer (LifeScan, Milpitas, CA) at 0, 15, 30, 60 and 120 minutes after injection.